The roles of cell adhesion molecules in tumor suppression and cell migration: A new paradox

In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.Key words: hepaCAM, cell adhesion molecules, tumor suppressor, migration, E-cadherin, CADM1, integrin α7, CEACAM1It is well known that many cell adhesion molecules function as tumor suppressors (reviewed in ref. ¹). These molecules exert their tumor suppressive effect mainly through cell-adhesion-mediated contact inhibition. Cell adhesion molecules allow cells to communicate with one another or to the extracellular environment by mediating cell-cell or cell-extracellular matrix (ECM) interactions (reviewed in refs. ² and ³). Broadly, these proteins can be classified into five families including immunoglobulin superfamily, integrins, cadherins, selectins and CD44. Apart from participating in the development and maintenance of tissue architecture, cell adhesion molecules serve as cell surface receptors critical for capturing, integrating and transmitting signals from the extracellular milieu to the cell interior (reviewed in refs. ² and ³). These signaling events are vital for the regulation of a wide variety of cellular functions including embryogenesis, immune and inflammatory responses, tissue repair, cell migration, differentiation, proliferation and apoptosis. Alterations of these cell adhesion molecules are a common event in cancer (reviewed in refs. ^{1, 2, 4} and ⁵). The disrupted cell-cell or cell-ECM adhesion significantly contributes to uncontrolled cell proliferation and progressive distortion of normal tissue architecture. More importantly, changes in cell adhesion molecules play a causal role in tumor dissemination. Loss of cell adhesion contacts allows malignant cells to detach and to escape from the primary mass. Gaining a more motile and invasive phenotype, these cells break down the ECM and eventually invade and metastasize to distal organs.Based on the above understanding, it is conventionally accepted that cell adhesion molecules with tumor suppressor activity, when expressed in cancer cells, are able to exert inhibitory effect on cell motility. The ability of cells in migration/motility is a prerequisite for cancer invasion and metastasis (reviewed in refs. ¹ and ⁵). Indeed, a number of cell adhesion molecule-tumor suppressors have been reported to be capable of reducing cell migration. The most classical example is E-cadherin, a calcium-dependent cell adhesion molecule. E-cadherin is expressed exclusively in epithelial cells and its expression is commonly suppressed in tumors of epithelial origins. The cytoplasmic domain of E-cadherin interacts with catenins to establish an intracellular linkage with the actin cytoskeleton (reviewed in ref. ⁶). The assembly of E-cadherin with the cytoskeleton via catenins at the sites of adherens junctions is important for the stabilization of cell-cell adhesions. Disruption of E-cadherin-mediated cell-cell adhesion, due to loss of expression or function of E-cadherin and/or catenins, is assocated with tumor development and progression (reviewed in ref. ⁷). Forced expression of E-cadherin in several cancer cell lines not only slows down cell growth^8,9 but also significantly reduces the invasiveness of the cells.^10,11 On the other hand, inhibition of E-cadherin by function-blocking antibodies and antisense RNA restores the invasiveness in non-invasive transformed cells.¹¹ Furthermore, using a transgenic mouse model of pancreatic beta-cell carcinogenesis, it has been demonstrated that E-cadherin-mediated cell adhesion is important in preventing the transition from well differentiated adenoma to invasive carcinoma.¹²Cell adhesion molecule 1 (CADM1), another example, has also been implicated in cancer progression. CADM1 is a member of the immunoglobulin superfamily and mediates cell-cell adhesion.¹³ The molecule associates with the actin cytoskeleton via the differentially expressed in adenocarcinoma of the lung (DAL1) protein; and the formation of CADM1-DAL1 complex is dependent on the integrity of actin cytoskeleton.¹⁴ Inactivation of the CADM1 and/or DAL1 gene usually through methylation has been reported in diverse human cancers.^15,16 A paper by Ito et al. showed that restoration of CADM1 expression in esophageal squamous cell carcinoma cells not only suppresses cell growth, but also retards cell motility and invasion.¹⁶In contrast to E-cadherin and CADM1, integrin α7 is a cell-ECM adhesion molecule which also possesses tumor suppressor activity. Ren et al. showed that integrin α7 gene is mutated in several human malignances; and the mutations are associated with an increase in cancer recurrence.¹⁷ Forced expression of integrin α7 in integrin α7-deficient leiomyosarcoma cells results in decreased colony formation and slower cell motility. Conversely, knockdown of integrin α7 in lung cancer cells expressing wild-type integrin α7 increases the colony number and cell motility rate. In addition, the researchers revealed that mice bearing xenograft tumors overexpressing integrin α7 have reduced tumor size with no obvious metastasis.In 2005, we first reported the identification of a cell adhesion molecule belonging to the immunoglobulin superfamily, designated as hepaCAM.¹⁸ To date, we have shown that the gene is frequently downregulated in a variety of human cancers.^18,19 Re-expression of hepaCAM in the hepatocellular carcinoma HepG2 cells¹⁸ and breast cancer MCF7 cells¹⁹ inhibits colony formation and retards cell proliferation. In addition, expression of hepaCAM in MCF7 cells results in cell cycle arrest at the G₂/M phase and cellular senescence. Concurrently, the expression of several senescence-associated proteins including p53, p21 and p27 is enhanced. Moreover, downregulation of p53 by p53-specific small interfering RNA in cells expressing hepaCAM clearly reduces p21 without changing p27 and alleviates senescence, indicating that hepaCAM induces senescence through a p53/p21-dependent pathway.¹⁹ Together, the data suggest that hepaCAM is a tumor suppressor. Interestingly, the expression of hepaCAM in both HepG2 and MCF7 cells stimulates both cell-ECM adhesion and cell migration.^18,20,21 The function of hepaCAM as a tumor suppressor in cell migration is contradictory to other cell adhesion molecule-tumor suppressors. Noteworthily, hepaCAM-mediated cell motility is evidenced by its direct interaction with the actin cytoskeleton.²¹Evidences are currently emerging to support the contradictory roles of cell adhesion molecules that both inhibit cell growth and promote cell motility when restored in cancer cells. In addition to hepaCAM, the immunoglobulin superfamily carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is implicated to function as a tumor suppressor and a metastasis promoter. The characteristics and functions of CEACAM1 have been demonstrated in individual reports. CEACAM1 is frequently downregulated or dysregulated in multiple human tumors,^22–25 and is capable of suppressing cell growth and inducing apoptosis.^26–28 Ebrahimnejad et al. demonstrated that exogenous expression of CEACAM1 enhances melanoma cell invasion and migration; and this enhanced motility can be reverted by anti-CEACAM antibodies.²⁹ The ability of CEACAM to co-stimulate tumor suppression and invasion was finally established by Liu et al. in restricting thyroid cancer growth but promoting invasiveness.³⁰ Introduction of CEACAM1 into CEACAM1-deficient thyroid cancer cells results in G₁/S phase cell cycle arrest accompanied by elevated p21 expression and diminished Rb phosphorylation. Overexpression of CEACAM1 also increases cell-ECM adhesion and promotes cell migration and tumor invasiveness. In xenografted mice, CEACAM1 expression results in reduced tumor growth but increased tumor invasiveness. Conversely, silencing of endogenous CEACAM1 accelerates tumor growth and suppresses invasiveness.³⁰It is an exciting issue to address why a cell adhesion molecule is able to suppress tumor growth yet promote tumor progression. Could there be a molecular switch that controls the functions of the gene between a tumor suppressor and a migration promoter in cancer or are the functions executed simultaneously? The expression level, the extracellular cues as well as the interacting partners of the cell adhesion molecules may likely play a critical role in regulating its functions. The question is under what circumstances these factors come into play. To answer all these questions, and maybe more, on the intriguing findings of these proteins, more extensive and intensive experimentation is required. Nevertheless, it is obvious that the emergence of these cell adhesion molecules that function in a contradictory manner opens a new chapter to the biological significance of cell adhesion molecules.     

Cell adhesion molecules in context: CAM function depends on the neighborhood

Cell adhesion molecules (CAMs) are now known to mediate much more than adhesion between cells and between cells and the extracellular matrix. Work by many researchers has illuminated their roles in modulating activation of molecules such as receptor tyrosine kinases, with subsequent effects on cell survival, migration and process extension. CAMs are also known to serve as substrates for proteases that can create diffusible fragments capable of signaling independently from the CAM. The diversity of interactions is further modulated by membrane rafts, which can co-localize or separate potential signaling partners to affect the likelihood of a given signaling pathway being activated. Given the ever-growing number of known CAMs and the fact that their heterophilic binding in cis or in trans can affect their interactions with other molecules, including membrane-bound receptors, one would predict a wide range of effects attributable to a particular CAM in a particular cell at a particular stage of development. The function(s) of a given CAM must therefore be considered in the context of the history of the cell expressing it and the repertoire of molecules expressed both by that cell and its neighbors.Key words: cell adhesion molecule, fibroblast growth factor receptor, epidermal growth factor receptor, L1, NCAM, Neuroglian, fasciclin, membrane raft, ankyrin, doublecortin, ezrin, radixin, moesinCell migration, axon extension and dendrite arborization are all essential processes in creating the complex neural architectures of the developing brain. A number of CAMs, including those of the immunoglobulin superfamily (IgCAMs), integrins and cadherins, are known to mediate signaling between cells and between cells and the extracellular matrix (ECM). Because the greatest amount of IgCAM research has focused on L1-CAM and NCAM and their invertebrate homologs Neuroglian and Fasciclin II, these molecules will be the primary focus of this Commentary & View. IgCAMs are so named because their extracellular domains contain immunoglobulin repeats (usually 5–6). The Ig repeats are usually followed by fibronectin type-3 (Fn III) domains (2–5) and either transmembrane plus cytoplasmic domains or a glycosyl-phosphatidylinositol (GPI) linkage (reviewed in refs. ^1–3). IgCAMs can bind homophilically and heterophilically via their Ig and/or Fn III domains to achieve cell-cell and cell-ECM adhesion, which can simply stabilize the architecture of neural tissue, but can also transmit information to the cell interior.⁴ For example, a number of IgCAMs are known to bind to the cytoskeleton via linker molecules including Ankyrin, Doublecortin and members of the Ezrin-Radixin-Moesin family (ERMs).^5–12 Some of these linking interactions are thought to allow engagement or disengagement of a molecular “clutch module” (reviewed in ref. ¹³) similar to coupling of integrins to F-actin flow via focal adhesion proteins and are believed to be important in growth cone function and synaptogenesis.^14,15 Work from these groups suggests these linker molecules are expressed in different developmental windows, so that ERMs and Doublecortin are important in L1-mediated neurite outgrowth and suppression of neurite branching, while subsequent Ankyrin expression and binding to L1 blocks outgrowth and fosters axon stabilization and synaptogenesis.^6–12 Another important example of outside-in signaling by IgCAMs is their ability, via Ankyrin''s multivalent binding sites, to cluster and position many receptors and channels at specific cellular locations such as axon initial segments and nodes of Ranvier (reviewed in ref. ¹² and ¹⁶). IgCAMs have also been shown to be substrates for matrix metalloproteases (MMPs), which can cleave extracellular domains, allowing the fragments to act as diffusable signaling molecules and changing the signaling effects of the remaining membrane-bound moieties.^17,18     

L1 cell adhesion molecules as regulators of tumor cell invasiveness

Fast growing malignant cancers represent a major therapeutic challenge. Basic cancer research has concentrated efforts to determine the mechanisms underlying cancer initiation and progression and reveal candidate targets for future therapeutic treatment of cancer patients. With known roles in fundamental processes required for proper development and function of the nervous system, L1-CAMs have been recently identified as key players in cancer biology. In particular L1 has been implicated in cancer invasiveness and metastasis, and has been pursued as a powerful prognostic factor, indicating poor outcome for patients. Interestingly, L1 has been shown to be important for the survival of cancer stem cells, which are thought to be the source of cancer recurrence. The newly recognized roles for L1CAMs in cancer prompt a search for alternative therapeutic approaches. Despite the promising advances in cancer basic research, a better understanding of the molecular mechanisms dictating L1-mediated signaling is needed for the development of effective therapeutic treatment for cancer patients.Key words: L1CAMs, cancer, metastasis, axon guidance, cancer stem cell, migration, invasionA major obstacle in oncology is the early diagnosis and curative therapeutic intervention of locally invasive cancers that rapidly disseminate from the primary tumor to form metastases. The standard treatment for malignant tumors consists of surgical removal of the tumor mass followed by chemo- and radiotherapy in order to eradicate the remaining cancer cells. Despite such aggressive intervention, a population of resistant cancer cells often remains intact and is thought to be the source of cancer recurrence.During the past decades, cancer basic research has focused on determining the molecular mechanisms underlying cancer initiation and progression that can provide a basis for the development of new and effective therapeutic treatments for cancer patients. An important finding was the discovery that cancer onset and development are often associated with alterations in the expression of cell adhesion molecules, which are likely to stimulate tumor cell invasiveness by signaling mechanisms that enhance cell migration.¹ The L1 family of neural cell adhesion molecules (L1-CAMs), which is comprised of four structurally related transmembrane proteins L1, CHL1, NrCAM and neurofascin (Fig. 1), is now in the spotlight of cancer research due to their upregulation in certain human tumors. L1-CAMs are transmembrane molecules of the immunoglobulin superfamily, characterized by an extracellular region of six immunoglobulin-like domains and four to five fibronectin type III repeats, followed by a highly conserved cytoplasmic domain, which is reversibly linked to the cell cytoskeleton through binding to ankyrin and ERM proteins (ezrin-radixin-moesin).² Its multi-domain structure allows complex heterophilic interactions with diverse cell receptors, although homophilic interactions also have a crucial role in L1-CAMs mediated signaling.Open in a separate windowFigure 1L1-CAMs: All have 6 Ig domains and 4–5 FN domains. The 186 kD Neurofascin isoform has a mucin-like Pro/Ala/Thr-rich (PAT) domain, while the 155 kD has only the 4 FN domains. RGD and DGEA motifs interact with integrins, while the FigQ/AY motif binds to ankyrin. ERM binding sites are indicated. The RSLE motif in L1 recruits AP2/clathrin adaptor for endocytosis.A wealth of studies has revealed L1-CAMs as pivotal components for proper development of the nervous system through regulation of cell-cell interactions. L1-CAMs have critical roles in neuronal migration and survival, axon outgrowth and fasciculation, synaptic plasticity and regeneration after trauma.² Neither CHL1 nor L1 is present on mature astrocytes, oligodendroglia or endothelial blood vessel cells in the brain, but CHL1 is upregulated in astrocytes upon injury³ and is present on oligodendroglial precursors.^4,5 During neural development, L1 plays an important role in the migration of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.⁶ In the cerebellum, L1 is required for the inward migration of granule neurons from the external granular layer and cooperates with NrCAM in regulating neuronal positioning.² Similarly, CHL1 controls area-specific migration and positioning of deep layer cortical neurons in the neocortex.⁷ In addition to its role in neuronal precursor positioning, L1 plays a crucial role in axon guidance, which is governed by repellent and attractive response mechanisms directed by Ephrins and Semaphorins and their receptors (Ephs, Neuropilins, Plexins).² The importance of L1-CAMs in the development and function of the nervous system is exemplified by developmental neuropsychiatric disorders that are associated with mutation or genetic polymorphisms in genes encoding L1 (X-linked mental retardation) and CHL1 (low IQ, speech and motor delay). Polymorphisms in L1 and CHL1 genes are also associated with schizophrenia, and NrCAM gene polymorphisms are linked to autism in some populations.²Recent studies have described upregulation of L1 in a variety of tumor types. Overexpression of L1 correlates with tumor progression and metastasis in certain human gliomas,⁸ melanoma,⁹ ovarian¹⁰ and colon carcinomas.^11–13 Interestingly, L1 was found to be present only in cells at the invasive front of colon cancers but not in the tumor mass.¹² L1 is also associated with micrometastasis to both lymph nodes and bone marrow in patients bearing other cancers, suggesting a potential role in early metastatic spread.¹¹ L1 has now been pursued as both a biomarker and a powerful prognostic factor, indicative of poor outcome for patients as observed for epithelial ovarian carcinoma¹⁰ and colorectal cancer.¹¹ More recently, L1 has been shown to be overexpressed in a small fraction of glioma cells, termed glioma stem cells, which are capable of self-renewal and generate the diverse cells that comprise the tumor.¹⁴ First characterized in acute myeloid leukemia,¹⁵ cancer stem cells have been recently described in a variety of solid tumors, including breast cancer, lung cancer and gastrointestinal tumors.¹⁶ In gliomas, L1 expression was shown to be required for maintaining the growth and survival of glioma stem cells.¹⁴ These findings suggest that L1 may be implicated not only in cancer invasiveness but also in cancer survival. It will be important to determine if L1 is also upregulated in other cancer stem cells as well as to define the role of L1-mediated signaling in other cancers. Although not extensively investigated, NrCAM has also been shown to be overexpressed in glioblastoma cell lines and several cases of high grade astrocytoma¹⁷ and ependymomas.¹⁸ Studies are needed to address whether CHL1 and neurofascin play analogous roles in cancer onset and progression.The molecular mechanisms of L1-mediated signaling that govern the migration of neuronal precursors and guidance of axons during the development of the nervous system may also be used by cancer cells to facilitate invasion and cancer progression. Integrins are well-characterized cooperative partners for L1-CAMs, and signal transduction pathways activated by this complex are known to promote cell adhesion and directional motility. L1/integrin-mediated signaling may converge with growth factor signaling networks to promote motility. Like L1, CHL1 cooperates with integrins to stimulate migration. All L1-CAMs reversibly engage the actin cytoskeleton through a conserved motif FigQ/AY in the cytoplasmic domain that contains a crucial tyrosine residue required for binding the spectrin adaptor ankyrin. Phosphorylation of the FigQY tyrosine decreases ankyrin binding, whereas dephosphorylation promotes L1-ankyrin interaction. Dynamic adhesive interactions controlled by phosphorylation/dephosphorylation of the ankyrin motif in L1 family members may enable a cell to cyclically attach and detach from the ECM substrate or from neighboring cells, thus facilitating migration.¹ Another way L1 promotes cell migration is by stimulating endocytosis of integrins, reducing cell adhesion to the extracellular matrix.¹⁹ Thus, it is reasonable to speculate that upregulation of L1 in cancer may result in increased L1-mediated signaling and, consequently, increased cell migration.L1-CAMs are cleaved by metalloproteases, releasing functionally active ectodomain fragments that are laid down as “tracks” on the extracellular matrix (ECM). These fragments can cause autocrine activation of signal transduction pathways, promoting cell migration through heterophilic binding to integrins.²⁰ Specifically, L1 is cleaved constitutively or inducibly by the ADAM family metalloproteases (a disintegrin and metalloprotease) ADAM10 and ADAM17, which stimulates cell migration and neurite outgrowth during brain development.^20,21 In colon cancer, L1 colocalizes with ADAM 10 at the invasive front of the tumor tissue, suggesting that L1 shedding may play a role in cancer invasiveness.¹² Similarly, CHL1 is shed by ADAM8, which was reported to promote cell migration and invasive activity of glioma cells in vitro and is highly expressed in human brain tumors including glioblastoma multiforme, correlating with invasiveness in vivo.²² Furthermore, NrCAM, found in pancreatic, renal and colon cancers, is subject to ectodomain shedding,²³ but its function in regulating cell migration or invasion has not yet been studied.Given the newly recognized roles of L1 in tumor progression, a growing body of experimental studies has explored novel therapeutic approaches targeting L1-CAMs. Antibody-based therapeutic strategies are being pursued to functionally inhibit homophilic and heterophilic interactions of cell adhesion molecules to suppress tumor invasive motility. L1 monoclonal antibodies reduce in vivo growth of human ovarian and colon carcinoma cells in mouse xenograft models.^13,24,25 L1 targeting using lentiviral-mediated short hairpin RNA (shRNA) interference decreases growth and survival of glioma stem cells in vitro, suppresses tumor growth, and increases survival of tumor-bearing animals.¹⁴ These findings raise the possibility that L1 represents a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. Cancer stem cells represent a potential target for future treatment of different cancer as these cells are believed to be responsible for cancer recurrence.²⁶ Promoting cancer stem cell differentiation by drug treatment could potentially reduce stem cells properties of self-renewal and proliferation, leading to inhibition of tumor growth.Inhibitors of metalloproteases that block L1-CAM shedding represent a potentially novel approach to curtailing tumor invasiveness. Chemical inhibitors of ADAMS are appealing for glioma therapy due to their diffusability, which circumvents blood-brain barrier limitations. Another novel approach involves the secreted axon repellent protein, Semaphorin 3A (Sema3A). L1-CAMs serve as co-receptors for Sema3A by cis binding in the plasma membrane to Neuropilin-1, important for repellent axon guidance.² Interestingly, Sema3A inhibits invasiveness of prostate cancer cells²⁷ and migration and spreading of breast cancer cells in in vitro assays,²⁸ and thus may also be mediated by L1-CAMs. Such an approach could be potentially useful in mitigating invasion of cancer cells in gliomas and other tumors that are known to express L1 and Neuropilins. However, effective strategies for some types of cancer can promote cancer progression in other types. For example, Sema3A has been shown to contribute to the progression of pancreatic cancer²⁹ and colon cancer.³⁰ Thus, it is imperative that the molecular mechanisms underlying L1-mediated signaling are understood in a tissue specific manner. Despite the promising advances in cancer basic research, much more research is needed to better design strategies for cancer therapy.     

Significance of microtubule catastrophes at focal adhesion sites

Directional cell migration requires cell polarization and asymmetric distribution of cell signaling. Focal adhesions and microtubules are two systems which are essential for these. It was shown that these two systems closely interact with each other. It is known that microtubule targeting stimulates focal adhesion dissociation. Our recent study shows that focal adhesions, in turn, specifically induce microtubule catastrophe via a biochemical mechanism. We were able to track down one of the focal adhesion proteins paxillin which is involved in this process. Paxillin phosphorylation was previously shown to be the key component in the regulation of focal adhesion assembly or disassembly. Since microtubule catastrophe dynamic differs at the leading edge and cell rear, similar to paxillin phosphorylation levels, we suggest a model connecting asymmetric distribution of focal adhesions and asymmetric distribution of microtubule catastrophes at adhesion sites as a feedback loop.Key words: microtubule catastrophe, focal adhesion, microtubules, paxillin, cell motilityCell migration is important for many biological processes. It requires organized asymmetric dynamics of focal adhesions (FAs), sites where cells interact with extra cellular matrix. FAs appear at the leading edge as small transient dot-like structures termed focal complexes (FXs).^1,2 FX assembly and disassembly is regulated by phosphorilation status of paxillin a major FX protein.^3,4 Most of FXs form and disassemble rapidly. However, some adhesions mature in a force-dependent manner, into larger late adhesions. This process, involves both an increase in size and change in molecular composition^3,5 and is accompanied by a reduction in local paxillin phosphorylation.⁴ Late adhesions are more stable, immobile and undergo forced disassembly by multiple microtubule targeting events⁶ only underneath the approaching cell body or transform into fibrillar adhesions by a Src-dependent mechanism.⁷Similarly to the leading edge, proper adhesion patterns at the cell rear are also essential. Most trailing adhesions are initiated in protrusions at the rear and flanks of the cell as FX rapidly mature in response to tension and transform into sliding trailing adhesions.⁸ The process of sliding is complex. While adhesion proteins coupled with the actin cytoskeleton can be translocated relative to substratum, those that are associated with the membrane are thought to undergo treadmilling within the adhesion site.^9,10 Treadmilling, which includes disassembly of adhesion proteins at the distal end and reassembly at the proximal end,¹⁰ is accompanied by fusion with new adhesions formed in front of the sliding one.⁶ Thus, despite a protein composition similar to late adhesions, sliding adhesions are more dynamic. Not surprisingly, sliding adhesions have high paxillin phosphorylation at the distal end of the adhesion site, indicating very dynamic assembly/disassembly rates.⁴Several mechanisms have been proposed for the regulation of adhesion turnover (reviewed in ref. ¹¹). However, these have not accounted for the observed asymmetry of adhesion turnover. Understanding this requires examining the connection with another asymmetric intracellular system, the microtubule network. This dynamic network closely interacts with FAs. Microtubules play an essential role in cell migration and polarized distribution of signals within the cell. Multiple microtubule targeting to FA leads to their disassembly both at the leading edge and at the cell rear.⁶Unlike microtubule growth in other cell regions, growth at its leading edge is persistent, characterized by short periods of shrinkage.⁸ Simultaneous observation of microtubules and FAs show that microtubules specifically target adhesion sites.¹² More detailed analysis of microtubule dynamics reveals that FAs are preferable sites for microtubule catastrophes.¹³ Although FAs cover only about 5% of cell area more than 40% of catastrophes occur at these sites. The likelihood of microtubule catastrophe is seven times higher when a microtubule grows through a FA rather than through an adhesion-free area¹³ and about 90% of microtubules approaching adhesion sites undergo catastrophe. Although most of the catastrophes occur at late adhesions, due to their increased stability and lifespan, there is no difference in efficiency of catastrophe induction between small focal complexes and large rigid late adhesions.¹³ As FX do not have dense adhesion or actin plaque, it is likely that microtubule catastrophe is triggered by a biochemical mechanism rather than mechanical rigidity. This is also supported by the fact that mechanical obstacles in a cell do not necessarily cause microtubule catastrophe.¹³At the cell rear, microtubule dynamics differ from those at the leading edge. Microtubules spend less time in a growing phase and more time in pauses and shrinkage.⁸ Polymerization and depolymerization occur within a very limited area close to the cell edge.⁸ Live-cell imaging of cells expressing both microtubule and focal adhesion markers show that this complex dynamic sequence often happens within a single sliding adhesion. Microtubules that are captured at the proximal end of adhesion undergo multiple repetitive catastrophes at the distal end (Fig. 1) accompanied by rescue at the capture site. Thus, the capture mechanism significantly increases the lifetime of a microtubule and ensures that repetitive catastrophes occur at the single adhesion. This scenario leads to high catastrophe frequency at the cell rear, resulting in intensive catastrophe-dependent regulation in this cell region.Open in a separate windowFigure 1Multiple microtubule catastrophes at the sliding adhesion. (A) Frame from TIRF video sequence of a fish fibroblast cell (CAR) co-transfected with GFP-tubulin (green) to visualize microtubules and Cherry-Zyxin (red) to mark focal adhesions. The boxed region is presented in the kymograph in (B). Bar, 10 µm. (B) Kymograph of microtubule dynamics at a trailing end focal adhesion. Top panel shows microtubule (MT) only. Bottom panel shows life history plot of MT (green line shows movement of MT end) in relation to focal adhesions (red). Arrows show catastrophes at the distal end of adhesion, arrowheads show capture at the proximal end of adhesion.Detailed analysis of microtubule catastrophe localization shows that they occur at the areas of FAs where paxillin is enriched and highly phosphorylated.^4,13 Paxillin was shown to interact with microtubules through its Lim2/Lim3 domain.¹⁴ Purified GST-Lim2/Lim3 fragment injected into the cell localizes to FAs, displacing endogenous paxillin.¹³ This leads to a 40% decrease in the number of microtubule catastrophe events at adhesion sites,¹³ indicating that paxillin is needed for catastrophe initiation.In summary, we conclude that microtubule catastrophes at focal adhesions are specific events that are triggered by a biochemical mechanism. This process involves the focal adhesion protein paxillin, which may serve as a docking site for microtubules and/or microtubule catastrophe factors. The nature of catastrophe factors remains to be clarified. Possible mechanisms include molecules which induce microtubule catastrophe directly, such as stathmin,¹⁵ or molecules which regulate catastrophe-inducing factors activity. Alternatively, catastrophe factors at adhesion sites could act by removing stabilizing factors from microtubule tips. Thus, allowing already active catastrophe-inducing molecules such as kinesin-13 family member MCAK^16,17 to complete their function. Furthermore, microtubule catastrophe at paxillin-enriched areas, followed by release of microtubule-associated factors, may be involved in paxillin phosphorylation. This local regulation of adhesion disassembly would close the feed-back loop to microtubule regulation of FA turnover.In this model, asymmetric distribution of microtubule catastrophes is tightly linked to asymmetric regulation of FA. Since asymmetric FA dynamics in a cell are critical for organization of the actin cytoskeleton, tensile force distribution and directional cell migration, we conclude that microtubule catastrophes serve as important regulatory events for asymmetric signaling and dynamics of the whole cell (Fig. 2).Open in a separate windowFigure 2Model for asymmetric focal adhesion and microtubule dynamics. Focal complexes at the leading edge either disassemble or mature in response to tension. Microtubules undergo catastrophe both at focal complexes and late adhesions. Late adhesions disassemble in response to multiple microtubule targeting. At the cell rear a microtubule is captured at the proximal end of sliding adhesion and undergoes multiple catastrophes at its distal end, supporting disassembly of this region.     

Cdk5: A regulator of epithelial cell adhesion and migration

Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.Key words: Cdk5, Src, Rho, stress fibers, epithelial cells, cell adhesion, cell migrationAnchoring of epithelial cells to their basement membrane is essential to maintain their morphology, normal physiological function and survival. Cells attach to extracellular matrix components by means of membrane-spanning integrins, which cluster and link to the actin cytoskeleton via components of focal adhesions. At focal adhesions, actin is bundled into stress fibers, multi-protein cellular contractile machines that strengthen attachment and provide traction during migration.¹ Stress fiber contraction is generated by myosin II, a hexamer containing one pair of each non-muscle heavy chains (NMHCs), essential light chains, and myosin regulatory light chains (MRLC). Myosin motor activity is regulated by phosphorylation of MRLC at Thr18/Ser19 and is required to generate tension on actin filaments and to maintain stress fibers.¹ Although a number of kinases have been identified which phosphorylate MRLC at Thr18/Ser19, the principal kinases in most cells are myosin light chain kinase (MLCK)² and Rho-kinase (ROCK),³ a downstream effector of the small GTPas, RhoA.Rho family small GTPases play a central role in regulating many aspects of cytoskeletal organization and contraction.⁴ These GTPases are subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1,^5,6 and negative regulation by GTPase-activating proteins (GAPs), such as p190RhoGAP.⁷ As cells spread, the Rho-family GTPase, Cdc42, is activated at the cell periphery, leading to the formation of numerous filapodia. Focal adhesion formation is first seen at the tips of these filapodia as focal adhesion proteins such as talin and focal adhesion kinase (FAK) bind to the intracellular domains of localized integrins.⁸ Src is recruited to activated FAK at the nascent focal adhesion and generates binding sites for additional focal adhesion proteins by phosphorylating FAK and paxillin.⁹ Src activity is essential for the further maturation of the focal adhesion and for activating the Rho-family GTPase, Rac, leading to Arp2/3-dependent actin polymerization, formation of a lamellipodium and extension of the cell boundary. Simultaneously, Src inhibits RhoA by phosphorylating and activating its upstream inhibitor, p190RhoGAP. As the focal adhesion matures, Src is deactivated, allowing the Rho activation necessary for mDia-dependent actin polymerization,¹⁰ myosin-dependent cytoskeletal contraction⁵ and tight adhesion to the extracellular matrix. Since new focal adhesions continually form at the distal boundary of the spreading cell, the most mature and highly contracted stress fibers are localized at the center of the cell.Cell adhesion is an essential component of cell migration: if adhesion is too weak, cells can not generate the traction necessary for migration; if it is too strong, they are unable to overcome the forces that anchor them in place. Thus, the relationship between adhesion force and migration rate is a bell-shaped curve.¹¹ Migration rate increases as adhesive strength increases until an optimum value is reached. Thereafter, increases in adhesive strength decrease migration rate. Since the strength of adhesion depends on extracellular matrix composition as well as the types of integrin expressed in the cell, a decrease in adhesive strength may result in either faster or slower cell migration.Several lines of evidence indicate that the proline-directed serine/threonine kinase cyclin dependent kinase 5 (Cdk5) plays an integral role in regulating cell adhesion and/or migration in epithelial cells.^12–17 Cdk5 is an atypical member of cyclin dependent kinase family, which is activated by the non-cyclin proteins, p35 or p39.¹⁸ Cdk5 is most abundant in neuronal cells where it also regulates migration and cytoskeletal dynamics.¹⁹ In neurons, Cdk5 exerts its effects on migration at least in part by phosphorylating FAK,¹⁹ and the LIS1 associated protein, NDEL1.²⁰ In contrast, recent findings have revealed two novel pathways involved in Cdk5-dependent regulation of migration in epithelial cells.^16,17One of these newly discovered mechanisms links Cdk5 activation to control of stress fiber contraction.¹⁶ We have found that Cdk5 and its activator, p35, co-localize with phosphorylated myosin regulatory light chain (MRLC) on centrally located stress fibers in spreading cells.¹⁶ Moreover, Cdk5 is strongly activated in spreading cells as central stress fiber contraction becomes pronounced.²¹ Since contraction of these central stress fibers is primarily responsible for tight attachment between the cell and the extracellular matrix,⁵ the above findings suggested that Cdk5 might regulate cell adhesion by regulating MRLC phosphorylation. To test this possibility we inhibited Cdk5 activity by several independent means and found that MRLC phosphorylation was likewise inhibited. In addition, we found that inhibiting Cdk5 either prevented the formation of central stress fibers or led to their dissolution. The concave cell boundaries characteristic of contracting cells were also lost.¹⁶ Since MRLC lacks a favorable site for phosphorylation by Cdk5, we asked whether Cdk5 might affect the upstream signaling pathways that regulate MRLC phosphorylation. Experiments with specific pathway inhibitors indicated that the MRLC phosphorylation involved in stress fiber contraction in lens epithelial cells was regulated largely by Rho-kinase (ROCK). Inhibiting Cdk5 activity not only significantly reduced ROCK activity, but also blocked activation of its upstream regulator, Rho. To explore the mechanism behind the Cdk5-dependent regulation of Rho, we turned our attention to p190RhoGAP, which appears to play a major role in regulating Rho-dependent stress fiber contraction.⁷ This RhoGAP must be phosphorylated by Src to be active; as a result, Rho activity is low in the early stages of cell spreading, when Src activity is high. At later times, Src activity falls, p190RhoGAP activity is lost, and Rho-GTP is formed, enabling Rho-dependent myosin phosphorylation and stress fiber contraction.^9,10 We have found that inhibiting Cdk5 activity during this later stage of cell spreading increases Src activity and Src-dependent phosphorylation of its substrate, p190RhoGAP. This in turn leads to decreased Rho activity accompanied by loss of Rho-dependent myosin phosphorylation, dissolution of central stress fibers, and loss of cell contraction (Fig. 1). Moreover, inhibiting Src protects cells from the loss of Rho activation and dissolution of central stress fibers produced by inhibiting Cdk5.¹⁶ Since the effects of Cdk5 on Rho-dependent cytoskeletal contraction appear to be mediated almost entirely through Cdk5-dependent regulation of Src, it will be particularly important to determine how Cdk5 limits Src activity.Open in a separate windowFigure 1Cdk5 inhibition reduces contraction of preformed stress fibers. (A) Cells were spread on fibronectin for 60 min to adhere, allowing them to form focal adhesion and stress fibers (pre-incubation) and then further incubated for 2 h in absence (control) or presence of Cdk5 inhibitor (olomoucine) and stained with phalloidin. The cells without olomoucine (control) had concave boundaries and well-formed stress fibers. Olomoucine treated cells showed loss of central stress fibers and failure to contract. Scale bar = 20 µ. (B) experimental conditions were same as shown in (A). Cdk5 inhibitor, olomoucine, was added after 1 h of spreading (indicated as t = 0) and cells were incubated for an additional 2h in absence or presence of olomoucine. Cell lysates were immunoblotted with antibodies for pMRLC (upper) and MRLC (middle). Tubulin was used as a loading control (lower). Lane 1: untreated (at 0 h); Lane 2: untreated (at 2 h); Lane 3: Cdk5 inhibitor (olomoucine) treated. (C) results of three independent experiments of the type shown in (B) were quantified by densitometry and normalized to determine the relative levels of pMRLC at each time. Statistical analysis demonstrated a significant (p < 0.05) decrease in pMRLC level in olomoucine treated cells compared to untreated cells.The central stress fibers regulated by Cdk5 play a central role in anchoring cells to the substratum, and their loss when Cdk5 is inhibited will reduce adhesion. As discussed above, reduction in cell adhesion may either increase or decrease the rate of cell migration, depending on the cell type and extracellular matrix composition. In lens and corneal epithelial cells, the reduction in adhesion produced by Cdk5 inhibition promotes cell migration.^13,15,16,22 Moreover, regulation of Rho/Rho-kinase signaling by Cdk5 seems to be a major factor in determining the migration rate, since inhibitors of Cdk5 and Rho-kinase increased lens epithelial cell migration rate equivalently and inhibiting both produced no additional effect.¹⁶Interestingly, an independent line of investigation has shown that this is not the only mechanism underlying Cdk5-dependent regulation of cell adhesion and migration. Cdk5 also localizes at focal contacts at the cell periphery and phosphorylates the focal adhesion protein talin.¹⁷ The talin phosphorylation site has been identified as S425, near the FERM domain in the talin head region. Upon focal adhesion disassembly, this region is separated from the talin rod domain by calpain-dependent cleavage.²³ Phosphorylation at S425 by Cdk5 blocks ubiquitylation and degradation of the talin head by inhibiting interaction with the E3 ligase, Smurf1. This leads, ultimately, to greater stability of lamellipodia and newly formed focal adhesions, thus strengthening adhesion to the substrate.¹⁷ Although the exact molecular events involved in this stabilization are not yet clear, it has been suggested that the talin head may “prime” integrins to bind full length talin.²⁴ One possible scenario describing how this might occur is shown in Figure 2. By permitting the isolated head region to escape degradation following calpain cleavage, Cdk5-dependent phosphorylation may stablize a pool of talin head domains to bind focal contacts within the lamellipodium. It is known that the isolated talin head region can bind and activate integrins during cell protrusion.²⁵ The resulting integrin activation would be expected to stabilize the lamellipodium by strengthening integrin-dependent adhesion. Since the head domain lacks sites for actin binding, which are located in the talin rod domain,²⁶ the bound head domain would have to be replaced by full length talin to enable focal adhesion attachment to the cytoskeleton.²⁵ The head domain might promote this replacement by recruiting the PIP-kinase needed to generate PI(4,5) P₂,²³ which facilitates binding of full length talin to integrin by exposing the auto-inhibited integrin binding sites.²⁷ The binding of full length talin and the resulting link between the integrins and the actin cytoskeleton would then further strengthen adhesion.²⁵ This model predicts that full length talin would bind poorly in the absence of Cdk5 activity, due to degradation of the talin head and the resulting limited availability of PI(4,5)P₂, and thus provides a possible explanation for the observed rapid turnover of peripheral focal adhesions.¹⁷ Clearly, other models may be proposed to explain the increase in adhesion produced by talin head phosphorylation, and deciding among them will be an active area for future investigation. Nonetheless, it is now certain that talin is a key substrate for Cdk5 at focal adhesions.Open in a separate windowFigure 2Mechanism of Cdk5-dependent regulation of cell adhesion and migration. Binding of p35 to Cdk5 forms the active Cdk5/p35 kinase, which regulates cell adhesion and migration in two distinct ways. Cdk5-dependent phosphorylation of the talin head domain at Ser425 prevents its ubiquitylation and degradation, allowing it to persist following calpain cleavage. The phosphorylated talin head may then bind to integrin at peripheral sites and recruit PIP-K, which converts PI(4)P to PI(4,5)P₂. PI(4,5)P₂ may promote replacement of the talin head by full length talin. Full length talin recruits other focal adhesion proteins to form the mature focal adhesion. The talin tail provides the site for the actin binding and polymerization. Polymerized actin is subsequently bundled into stress fibers. Cdk5/p35 also regulates the Rho-dependent myosin phosphorylation necessary for stress fiber stability and cytoskeletal contraction by limiting Src activity. This in turn decreases Src-dependent phosphorylation of p190RhoGAP, favoring Rho-GTP formation, Rho-dependent stress fiber polymerization, stabilization and contraction. Both pathways modulate cell migration by increasing adhesive strength.In summary, the presently available data indicate that Cdk5 has at least two distinct functions in cell adhesion (Fig. 2). On the one hand, it stabilizes peripheral focal adhesions and promotes their attachment to the cytoskeleton by phosphorylating the talin head. On the other hand, once the actin cytoskeleton has been organized into stress fibers, Cdk5 enhances the Rho activation essential for stability and contraction of central stress fibers by limiting Src activity. The discovery that Cdk5 is involved in two separate events required for efficient migration, suggests that it may coordinate multiple signaling pathways. The known involvement of Cdk5 and its activator, p35, in regulating microtubule stability suggests yet another mechanism by which Cdk5 activity may regulate cytoskeletal function. Microtubules are closely associated with stress fibers²⁸ and their depolymerization has been shown to release the Rho activating protein, GEF-H1, leading to Rho activation and Rho-dependent myosin contraction.⁶ Since cell adhesion and migration play an important role in the progression of many pathological conditions, Cdk5, its substrates and its downstream effectors involved in cell adhesion may provide novel targets for therapeutic intervention.^15,29     

A novel role for the Ste20 kinase SLK in adhesion signaling and cell migration

With over 60 members, the Sterile 20 family of kinases has been implicated in numerous biological processes, including growth, survival, apoptosis and cell migration. Recently, we have shown that, in addition to cell death, the Ste20-like kinase SLK is required for efficient cell migration in fibroblasts. We have observed that SLK is involved in cell motility through its effect on actin reorganization and microtubule-induced focal adhesion turnover. Scratch wounding of confluent monolayers results in SLK activation. The induction of SLK kinase activity requires the scaffold FAK and a MAPK-dependent pathway. However, its recruitment to the leading edge of migrating fibroblasts requires the activity of the Src family kinases. Since SLK is microtubule-associated, it may represent one of the signals delivered to focal contacts that induces adhesions turnover. A speculative model is proposed to illustrate the mechanism of SLK activation and recruitment at the leading edge of migrating cells.Key words: cell migration, cell adhesion, SLK, microtubules, adhesion turnoverCell migration is involved in multiple biological processes such as development, tissue regeneration, immune surveillance and tumor metastasis. Numerous studies reported a multitude of cellular and molecular players that take part in the signaling networks that regulate cell migration.^1,2 Recently, we reported the participation of a new member, the Ste20 serine/threonine kinase SLK, in the regulation of cell migration. We have shown that SLK is a novel adhesion disassembly signal that is activated and recruited downstream of the FAK/Src complex following scratch wound-induced migration.³ Furthermore, SLK-dependent signals are required to mediate microtubule-dependent focal adhesion tunrnover.³ These findings provide new insights into the mechanisms of cell migration and adhesion dynamics.Since sterile 20 protein (Ste20p) acts as a MAP4K in yeast, it was suggested that mammalian homologues of Ste20p also function as MAP4K.⁴ Several members of the Ste20 family of kinases have been identified in mammals and implicated in various biological processes such as stress responses, cell death and cytoskeletal reorganization.⁵ We and others previously identified a novel Ste20-related kinase termed SLK, which is a part of a signaling pathway mediating c-Jun terminal kinase 1 (JNK1) activation and apoptosis in cultured fibroblasts.^6–8 In addition, recent reports showed that SLK is involved in C2C12 myoblast differentiation and plays a role in cell cycle progression.^9,10 SLK is ubiquitously expressed, but during embryogenesis it is highly enriched in muscle and neuronal tissues.¹¹ It has been shown that SLK is associated with the microtubule cytoskeleton and we have demonstrated that SLK-induced disassembly of actin stress fibers can be inhibited by dominant negative Rac1.^12–14Recently, SLK was identified as a member of a new signaling pathway that induce vasodilatation in response to angiotensin II type 2 receptor activation.¹⁵ It was reported that SLK negatively regulates RhoA-dependent functions by phosphorylation of RhoA at Ser188.¹⁵ These findings suggest that SLK represents a novel relaxation signal involved in cytoskeletal remodeling and cell migration.We have observed that SLK is recruited to the leading edge of migrating fibroblasts by a mechanism involving c-Src signaling.³ The molecular mechanism regulating SLK recruitment is still unclear but is likely to implicate the association of SLK with another protein. The translocation of SLK could involve a microtubule-dependent mechanism leading to its redistribution to peripheral adhesions, using actin stress fibers as tracks. The Rho GTPases have been shown to be important in the targeting of signaling components, such as c-Src, to specific adhesion sites.^16,17 Whether SLK recruitment to the leading edge requires the Rho GTPases remains to be investigated. The Rho-mDia pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and is responsible for delivering APC/Cdc42 and c-Src to their respective sites of action.¹⁸ One attractive possibility is that mDia facilitates SLK-microtubule translocation in a c-Src dependent manner.Integrin molecules which link the extracellular matrix to the intracellular machinery are key players in initiating polarized cell migration into the wound. We investigated SLK activity in a scratch-induced migration model and have been able to decipher various signaling components regulating SLK activation.³ Using knockdown and dominant negative approaches, we showed that SLK is required for microtubule-dependent focal adhesion turnover and cell migration downstream of the FAK/Src complex.³The molecular mechanisms by which microtubules contribute to cell migration have been intensively studied. Geiger''s group provided the first demonstration that cytoskeletal modulation, such as microtubule disruption, triggers integrin-dependent signaling in the absence of external growth factor stimulation.¹⁹ The authors suggested that the involvement of microtubules in adhesion dependent signaling is related to microtubule interaction with the contractile actin-myosin system.¹⁹ By using a nocodazole washout system, it was shown that FAK and the GTPase dynamin are required for microtubule-induced focal adhesion disassembly.²⁰Adhesion turnover involves a number of adapters and signaling molecules, most of which are engaged in FAK signaling pathways.²¹ FAK stimulates adhesion disassembly through a signaling pathway that includes extracellular signal-regulated kinase (ERK) and myosin light chain kinase (MLCK).²² Our data have shown that SLK is activated downstream of FAK/Src/MAPK signaling, suggesting that SLK may be a new target of this pathway that leads to adhesion disassembly. Furthermore, if RhoA is a bona fide substrate for SLK in fibroblasts, then by phosphorylating and inhibiting RhoA, SLK could tilt the Rho/Rac antagonistic interplay toward relaxation and adhesion disassembly. Downstream targets of FAK and Src kinase activity often regulate the recruitment of adapter and structural protein complexes to adhesions.²² The integration of molecules such as zyxin, α-actinin or paxillin into focal contacts can lead to their stabilization and maturation into focal adhesions.²² Interestingly, depending on their phosphorylation state, these components can promote adhesion destabilization and turnover. Therefore, it is tempting to speculate that activated SLK at the leading edge may phosphorylate key signaling components to induce adhesion turnover.A recent study has shown that the frequency of microtubule catastrophes is higher at focal adhesion sites and this event leads to a local release of microtubule regulatory proteins, such as GEF-H1 and APC.²³ Signaling molecules that are released from the microtubules at adhesions could directly associate with molecular factors concentrated at the adhesion plaques, such as Src, PAK and Arp2/3. Furthermore, it was speculated that microtubule catastrophe could be associated with phosphorylated paxillin-dependent protein complexes.²³ One possibility is that through the microtubule, SLK is delivered to focal contacts or adhesions where it serves as a scaffold for disassembling signals. Alternatively, SLK may be phsophorylating key signaling molecules, which ultimately leads to adhesion destabilization and turnover.Overall, our recent findings suggest that SLK is novel regulator of focal adhesion turnover and cell migration (Fig. 1). The molecular mechanisms regulating SLK activity and SLK-dependent adhesion turnover remain to be uncovered and await the identification of SLK substrates.Open in a separate windowFigure 1Model for SLK activation and recruitment at the leading edge. A proportion of SLK is microtubule-associated, likely through a microtubule-binding protein (X). Following activation of the FAK/c-Src complex, signaling through the MAPK pathway can activate and recruit the microtubule-SLK complex, inducing adhesion turnover by destabilization of the actin network or focal contacts/adhesions through an unknown mechanism. (C) denotes a cargo protein coupling the microtubule to polymerized actin. Nocodazole treatment fails to recruit SLK resulting in stable adhesions.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth

Filopodia are key structures within many cells that serve as sensors constantly probing the local environment. Although filopodia are involved in a number of different cellular processes, their function in migration is often analyzed with special focus on early processes of filopodia formation and the elucidation of filopodia molecular architecture. An increasing number of publications now describe the entire life cycle of filopodia, with analyses from the initial establishment of stable filopodium-substrate adhesion to their final integration into the approaching lamellipodium. We and others can now show the structural and functional dependence of lamellipodial focal adhesions as well as of force generation and transmission on filopodial focal complexes and filopodial actin bundles. These results were made possible by new high resolution imaging techniques as well as by recently developed elastomeric substrates and theoretical models. The data additionally provide strong evidence that formation of new filopodia depends on previously existing filopodia through a repetitive filopodial elongation of the stably adhered filopodial tips. In this commentary we therefore hypothesize a highly coordinated mechanism that regulates filopodia formation, adhesion, protein composition and force generation in a filopodia dependent step by step process.Key words: filopodia, focal adhesion, cell force, filopodial focal complex, actinCell protrusion depends on collaborative interactions of lamellipodia and filopodia.¹ Although filopodia cannot drive cell migration alone, in contrast to lamellipodia, they are essential for many cell biological functions such as guidance of neuronal growth cones² or during angiogenesis.³ Furthermore, filopodia are vital to cell-cell contact establishment as described for epithelial cells⁴ or during dorsal closure in Drosophila,⁵ and are also implicated in cancer cell metastasis.^6,7 Lamellipodia as well as filopodia can be formed independently from each other,⁸ and recent results implicate independent basic mechanisms of cytoskeletal regulation for their formation. While lamellipodia protrusion is a well accepted Arp2/3-dependent process where actin branches constantly form the protrusive force at the leading edge of the lamella,⁹ the details of filopodia formation are far from being understood.^10–13 Although earlier experiments indicated Arp2/3 was also involved in filopodia formation,¹⁴ recent results point to a machinery that is far less dependent, or even possibly independent, of Arp2/3 with formins being the central regulating molecules instead.⁸As soon as filopodia start to form, they constantly sense their environment upon elongation. Transmembrane proteins such as cadherins or integrins^15,16 connect filopodia to surrounding cells, extracellular matrix, or even pathogens to form stable contacts. When filopodial adhesion fails, retraction takes place.¹⁷ Although integrins and talin have been shown to be initially present at these sites in an un-clustered but active state, many additional adhesion proteins take part in filopodial focal complexes (filopodial FXs).^16,18 Starting from a small VASP-containing adhesion spot at the tip of filopodia, proteins such as vinculin, paxillin, talin, tensin and even zyxin form an elongated filopodial FX behind the VASP spot along the filopodium. While integrin as well as VASP transport along the filopodia shaft via myosin-X has been described,¹⁹ it is still unclear whether additional adhesion proteins are also actively transported or whether diffusion takes place. Diffusion is typically a non-limiting process during cytoplasmic protein complex formation. However for filopodia, diffusion could have an important regulatory function as already hypothesized in theoretical models,²⁰ because they are small in width and densely filled with actin filaments. Therefore, local concentrations of soluble adhesion molecules might drop within filopodia upon FX formation resulting in a pure physical regulation of filopodial length as well as filopodial FX size.The almost complete focal adhesion site specific protein inventory of filopodia FXs^16,18 as indicated above provided further indications for a dependency of lamellipodial focal adhesions (FAs) on filopodial FXs. This hypothesis was confirmed using fluorescent live cell imaging to identify the transition of filopodial FXs into fully assembled FAs upon FX contact with the leading edge of the lamellipodium. While filopodial FXs were responsible for only a sub-fraction of FAs in fish fibroblasts,¹⁸ stable FAs of human keratinocytes were formed almost exclusively by enlargement of existing filopodial FXs¹⁶ (see scheme, Fig. 1).Open in a separate windowFigure 1Filopodia determine the fate of lamellipodial structures. Filopodia are formed by actin polymerization at their tip. Upon stable adhesion, a small but fully assembled filopodial focal complex (FX) is formed. This FX becomes enlarged in size upon lamellipodial contact to form focal adhesions. In parallel, the filopodial actin cross-linker fascin becomes exchanged by palladin and α-actinin as soon as the filopodial actin bundles are incorporated into the lamellipodium. In a next step, α-actinin becomes partially exchanged by myosin II, leading to enhanced force values applied at filopodial-originated FA sites bound to the substrate. The tight interaction between FAs and filopodial actin bundles reduces the actin retrograde flow within the filopodium in front of the FA (lower inlay) compared to filopodia lacking stable FAs in the lamellipodium (not shown). Adhesion sites formed in the lamellipodium lack connections to distinct actin bundles leading to low force application at these sites and short lifetimes (upper inlay).The structural dependency of lamellipodial complexes on filopodial protein aggregates could be also shown for actin bundles. Here, parallel oriented actin filaments become cross-linked by proteins such as fascin or IRSp53-Eps8-complex upon filopodia formation.^21,22 These tightly packed bundles of 15–30 single actin filaments originate from the lamellipodial actin meshwork.²³ Interestingly, filopodial actin bundles in turn also affect lamellipodial actin structures independent of whether the filopodium adheres in a stable manner or looses contact. Nemethova et al.¹⁸ described the contribution of non-adhering filopodia to the construction of concave bundles of actin filaments within the lamellipodium of fish fibroblasts. These bundles often extended in length and interconnected with adjacent bundles. Similar observations were found for fibroblasts of chicken embryos and neuronal growth cones.^24,25 Here, filopodial actin bundles were clearly shown to be the origin of nearly 85% of all actin bundles found in the lamella. These actin filaments typically pointed towards the direction of migration. Additionally, myosin II was associated with these filopodial derived actin filaments to form polarized actin bundles. Of equal importance are findings presented by Schäfer et al. in this issue. The authors analyzed the fate of stably adhered filopodia and identified a stepwise exchange of filopodial fascin against the actin cross-linker proteins palladin and especially α-actinin in areas where filopodia were just overgrown by the lamellipodial leading edge (schematically presented in Fig. 1). α-Actinin further induced incorporation of myosin II into filopodial actin bundles in the lamellipodium. The authors additionally found that FAs displayed an enhanced lifetime when adhered to these myosin containing actin filaments. Therefore, these findings could also explain the unusual stability of filopodial actin filaments in neuronal growth cones observed by Mallavarapu and Mitchison.¹⁷ For keratinocytes, filopodia-dependent actin bundles are the only myosin containing actin structures oriented in the direction of movement within the lamellipodium and the lamella. Sensitivity and resolution improvements in cell force analyses further proved that these actin bundles were responsible for almost the entire force transmitted from the lamellipodium of migrating keratinocytes to the substrate. These forces were transferred at FA sites emerging from filopodial FXs, proving the importance of filopodia in lamellipodial structures and functions. Although filopodia-independent adhesion sites are also formed in keratinocytes right behind the leading edge, these sites are neither connected to detectable actin filament bundles nor do they transmit significant forces (see scheme, Fig. 1). Consequently, their sizes and life spans are strongly reduced (Schäfer et al., this issue).Recent results in keratinocytes additionally close the circle from stably adhered filopodia to the generation of new ones. Our original observations indicated that new filopodia were mainly formed in a direct extension of focal adhesions. Since these adhesion sites also depended on previously adhered filopodial FXs, a closer look revealed a consecutive outgrowth of the same filopodia.¹⁶ These cycles were only interrupted when outgrowing filopodia did not adhere in a stable manner between outgrowth cycles. Present results suggest that the same tip complex is present in all subsequently formed filopodia with a VASP tip signal remaining in place during successive filopodial elongations. As a result, well aligned, consecutive elongated focal adhesions can be found in keratinocytes. We can only speculate whether such an Arp2/3-independent mechanism describes a basic principle in filopodia formation at this point, but similar results have been observed for fish fibroblasts with a repetitive and alternating transition between filopodia and microspikes as filopodia-like structures barely extending over the lamellipodial leading edge.¹⁸The strong interdependency between lamellipodial FAs and stably adhered filopodia is also highlighted by actin retrograde flow analyses in keratinocytes (Schäfer et al. this issue). Retrograde actin flow is generated by actin polymerization at the cell front and myosin activity pulling the filaments rearwards. The interaction of actin with FAs is known to dampen flow rates in front of lamellipodial FAs.²⁶ Furthermore, filamentous-actin dynamics measured in lung epithelial cells showed a fast retrograde actin flow at the leading edge compared to rates within the lamellae. The highest flow rates were in the range of 0.3–0.5 µm/min.²⁷ Interestingly, keratocytes exhibited ten times slower flow rates at the leading edge,²⁸ indicating that retrograde flow strongly depends on the cell type analyzed. Actin filaments polymerizing at the tips of filopodia also undergo retrograde flow, but these flow rates are much faster compared to those found in lamellipodia,²⁴ as shown by bleaching experiments in chick embryo fibroblasts with flow rates approximately two-fold faster in filaments derived from filopodia compared to flow rates measured within the lamellipodium. These flow rates of approximately 1.3 µm/min were similar to those found for filopodia in other studies.²² Furthermore, we could show that this retrograde flow rate strongly depends on stable FAs formed behind the filopodium (Schäfer et al. this issue and Fig. 1). In the absence of these FAs, actin retrograde flow is doubled once more to rates of approximately 2.5 µm/min in filopodia. Therefore, although rates of FAs containing filopodia are still much higher than those found in lamellipodia, these rates are still slowed down indicating an effective connection between FAs and filopodial actin. These results further imply that myosin II incorporation into filopodial-originated actin bundles is responsible for enhanced retrograde flow rates in filopodia compared to rates found in the lamellipodium and that myosin II incorporation does not depend on stably adhered FAs directly behind filopodia. These data also strongly support the hypothesis that new filopodia form in front of stable lamellipodial FAs. It will be an intriguing question for future studies to analyze whether the reduced retrograde flow speeds in front of lamellipodial FAs might even be a prerequisite for efficient assembly and stable adhesion of small filopodial FXs, or perhaps even for filopodia formation in general.Taking into account all the currently known functions of filopodia, the presented results finally indicate that filopodia might be characterized best not only by one but actually two main functions. The first function is environmental sensing. Various transmembrane proteins can be involved leading to various roles for filopodia such as formation of cell-cell or cell-matrix interactions.^5,15 Although these functions in environmental sensing seem to be highly diverse, force generation along filopodial-originated actin bundles as the second function for filopodia might be of universal importance independent of the cell type that forms them. Force transmission along cell-pathogen interacting filopodia have been observed,²⁹ and the formation of adherens junctions after filopodia mediated cell-cell interaction is also a cell force dependent process.⁵ Therefore, these observations fit well to the currently presented data by Schäfer et al. (this issue) proving the importance of filopodia-dependent cell matrix interactions in cell force generation in the direction of migration (see scheme, Fig. 1).Present in almost every moving cell type, filopodia are therefore much more than just sensors for environmental conditions. In fact, these needle-like structures are the starting point for essential structures of adhesion and movement. Independent of whether they adhere stably or not, filopodia define the position of cellular adhesion sites, actin bundles, cell force generation and application, and, finally, the new filopodia to be formed.     

The TRPA1 channel and oral hypoglycemic agents

Diabetes mellitus type 2 (DM2) results from the combination of insulin unresponsiveness in target tissues and the failure of pancreatic β cells to secrete enough insulin.¹ It is a highly prevalent chronic disease that is aggravated with time, leading to major complications, such as cardiovascular disease and peripheral and ocular neuropathies.² Interestingly, therapies to improve glucose homeostasis in diabetic patients usually involve the use of glibenclamide, an oral hypoglycemic drug that blocks ATP-sensitive K⁺ channels (K_ATP),^3,4 forcing β cells to release more insulin to overcome peripheral insulin resistance. However, sulfonylureas are ineffective for long-term treatments and ultimately result in the administration of insulin to control glucose levels.⁵ The mechanisms underlying β-cell failure to respond effectively with glibenclamide after long-term treatments still needs clarification. A recent study demonstrating that this drug activates TRPA1,⁶ a member of the Transient Receptor Potential (TRP) family of ion channels and a functional protein in insulin secreting cells,^7,8 has highlighted a possible role for TRPA1 as a potential mediator of sulfonylurea-induced toxicity.     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Matrix metalloproteinase (MMP) and TGFβ1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.Key words: wound healing, migration, matrix metalloproteinase, transforming growth factor, skin, corneaWound healing is a well-ordered but complex process involving many cellular activities including inflammation, growth factor or cytokine secretion, cell migration and proliferation. Migration of skin keratinocytes and corneal epithelial cells requires the coordinated expression of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), small GTPases, and macrophage stimulating protein (reviewed in refs. ¹ and ²). The epithelial cells in turn regulate the expression of matrix metalloproteinases (MMPs), extracellular matrix (ECM) proteins and integrins during cell migration.^1,3,4 TGF-β is a well-known cytokine involved in processes such as cell growth inhibition, embryogenesis, morphogenesis, tumorigenesis, differentiation, wound healing, senescence and apoptosis (reviewed in refs. ⁵ and ⁶). It is also one of the most important cytokines responsible for promoting the migration of skin keratinocytes and corneal epithelial cells.^3,6,7TGFβ has two quite different effects on skin keratinocytes: it suppresses their multiplication and promotes their migration. The TGFβ-induced cell growth inhibition is usually mediated by Smad signaling, which upregulates expression of the cell cycle inhibitor p21^WAF1/Cip1 or p12^CDK2-AP1 in HaCaT skin keratinocyte cells and human primary foreskin keratinocytes.^8,9 Keratinocyte migration in wounded skin is associated with strong expression of TGFβ and MMPs,¹ and TGFβ stimulates the migration of manually scratched wounded HaCaT cells.¹⁰ TGFβ also induces cell migration and inhibits proliferation of injured corneal epithelial cells, whereas it stimulates proliferation of normal corneal epithelial cells via effects on the MAPK family and Smad signaling.^2,7 Indeed, skin keratinocytes and corneal epithelial cells display the same two physiological responses to TGFβ during wound healing; cell migration and growth inhibition. However as mentioned above, TGFβ has a different effect on normal cells. For example, it induces the epithelial to mesenchymal transition (EMT) of normal mammary cells and lens epithelial cells.^11,12 It also promotes the differentiation of corneal epithelial cells, and induces the fibrosis of various tissues.^2,6The MMPs are a family of structurally related zinc-dependent endopeptidases that are secreted into the extracellular environment.¹³ Members of the MMP family have been classified into gelatinases, stromelysins, collagenases and membrane type-MMPs (MT-MMPs) depending on their substrate specificity and structural properties. Like TGFβ, MMPs influence normal physiological processes including wound healing, tissue remodeling, angiogenesis and embryonic development, as well as pathological conditions such as rheumatoid arthritis, atherosclerosis and tumor invasion.^13,14The expression patterns of MMPs during skin and cornea wound healing are well studied. In rats, MMP-2, -3, -9, -11, -13 and -14 are expressed,¹⁵ and in mice, MMP-1, -2, -3, -9, -10 and -14 are expressed during skin wound healing.¹ MMP-1, -3, -7 and -12 are increased in corneal epithelial cells during Wnt 7a-induced rat cornea wound healing.¹⁶ Wound repair after excimer laser keratectomy is characterized by increased expression of MMP-1, -2, -3 and -9 in the rabbit cornea, and MMP-2, -9 in the rat cornea.^17,18 The expression of MMP-2 and -9 during skin keratinocyte and corneal epithelial cell migration has been the most thoroughly investigated, and it has been shown that their expression generally depends on the activity of MMP-14. MMP-14 (MT1-MMP) is constitutively anchored to the cell membrane; it activates other MMPs such as MMP-2, and also cleaves various types of ECM molecules including collagens, laminins, fibronectin as well as its ligands, the integrins.¹³ The latent forms of some cytokines are also cleaved and activated by MMP-14.¹⁹ Overexpression of MMP-14 protein was found to stimulate HT1080 human fibrosarcoma cell migration.²⁰ In contrast, the attenuation of MMP-14 expression using siRNA method decreased fibroblast invasiveness,²¹ angiogenesis of human microvascular endothelial cells,²² and human skin keratinocyte migration.¹⁰ The latter effect was shown to result from lowering MMP-9 expression. Other studies have shown that EGF has a critical role in MMP-9 expression during keratinocyte tumorigenesis and migration.^23,24 On the other hand, TGFβ modulates MMP-9 production through the Ras/MAPK pathway in transformed mouse keratinocytes and NFκB induces cell migration by binding to the MMP-9 promoter in human skin primary cultures.^25,26 Enhanced levels of pro-MMP-9 and active MMP-9 have also been noted in scratched corneal epithelia of diabetic rats.²⁷There is evidence that MMP-14 activates a number of intracellular signaling pathways including the MAPK family pathway, focal adhesion kinase (FAK), Src family, Rac and CD44, during cell migration and tumor invasion.^19,20,28 In COS-7 cells, ERK activation is stimulated by overexpression of MMP-14 and is essential for cell migration.²⁹ These observations all indicate that MMP-14 plays an important role in cell migration, not only by regulating the activity or expression of downstream MMPs but also by processing and activating migration-associated molecules such as integrins, ECMs and a variety of intracellular signaling pathays.³⁰Cell migration during wound healing is a remarkably complex phenomenon. TGFβ is just one small component of the overall process of wound healing and yet it triggers a multitude of reactions needed for cell migration. It is important to know what kinds of molecules are expressed when cell migration is initiated, but it is equally important to investigate the roles of these molecules and how their expression is regulated. Despite the availability of some information about how MMPs and signaling molecules can influence each other, much remains to be discovered in this area. It will be especially important to clarify how MMP-14 influences other signaling pathways since its role in cell migration is not restricted to digesting ECM molecules but also includes direct or indirect activation of cellular signaling pathways.     

Interplay between adhesion turnover and cytoskeleton dynamics in the control of growth cone migration

The migration of neuronal growth cones, driving axon extension, is a fascinating process which has been subject of intense investigation over several decades. Many of the key underlying molecules, in particular adhesion proteins at the cell membrane which allow for target recognition and binding, and cytoskeleton filaments and motors which power locomotion have been identified. However, the precise mechanisms by which growth cones coordinate, in time and space, the transmission of forces generated by the cytoskeleton to the turnover of adhesion proteins are still partly unresolved. To get a better grasp at these processes, we put here in relation the turnover rate of ligand/receptor adhesions and the degree of mechanical coupling between cell adhesion receptors and the actin rearward flow. These parameters were obtained recently for N-cadherin and IgCAM based adhesions using ligand-coated microspheres in combination with optical tweezers and photo-bleaching experiments. We show that the speed of growth cone migration requires both a fairly rapid adhesion dynamics and a strong physical connection between adhesive sites and the cytoskeleton.Key words: actin retrograde flow, molecular clutch, myosin, N-cadherin, IgCAMGrowth cones are motile structures at the distal extremity of axons responsible for pathfinding and neurite extension during nervous system development and repair (Fig. 1A). Growth cone advance relies on two coupled processes. First, an internal dynamics of the cytoskeletal network, with actin polymerization occurring at the leading edge, depolymerization in the central region, and myosin activity pulling on lamellipodial actin filaments.¹ These integrated mechanisms altogether result in a continuous retrograde flow of actin (Fig. 1B). This flow provides the mechanical tension that drives axonal extension, through a connection to the dynamic array of microtubules that fills the axon and invades the growth cone central domain.² Second, there is repeated formation and dissociation of transient contacts between growth cones and the extracellular matrix or adjacent cells. These contacts are mediated by trans-membrane cell adhesion molecules (CAMs), e.g. integrins,³ immunoglobulin CAMs (IgCAMs)^4,5 and cadherins,^6,7 which form specific ligand/receptor bonds with variable lifetimes. A still open question is how these two processes, i.e. actin flow and adhesion dynamic, are coordinated at the growth cone level and contribute to set migration speed. A thorough understanding of these mechanisms is important both from a fundamental perspective and for the design of new compounds to foster axon regeneration after injury.Open in a separate windowFigure 1Growth cone advance and actin flow. (A) Growth cone from a 2 DIV rat hippocampal neuron plated on N-cadherin coated glass. This growth cone moved forward at a speed of about 1 µm/min (B) Raw fluorescence image of transfected actin-GFP. (C) Sequential actin-GFP images were subtracted, giving rise to intensity variations that display the movement of newly assembled actin (black). Note the rapid retrograde movement of actin spots (arrowheads), at a velocity of several µm/min.The coupling between actin-based motility and substrate adhesion has been shown for certain adhesion molecules such as NCAM and N-cadherin to involve a “molecular clutch” (Fig. 2). This mechanism implies a direct transmission of traction forces from the cytoskeleton to the substrate through a strong physical connection between the actin flow and ligand-bound adhesion receptors.^8,9 The connection is likely provided by adaptor proteins that can make transient bridges between actin filaments and the cytoplasmic domain of adhesion molecules, i.e. α-and β-catenin in the case of N-cadherin,¹⁰ ankyrin and ezrin in the case of IgCAMs such as L1.^11–14 These purely mechanical connections can also be accompanied by signalling events such as Rac-1 activation by N-cadherin liganding¹⁵ and phosphorylation of the L1 intracellular tail that regulates binding to ankyrin.^11,12 When only few molecular bonds are formed, e.g. at low ligand density, coupling to the actin flow is not strong enough, resulting in “slippage.” In this process, transient bonds can be formed and broken repeatedly between ligand-occupied adhesion receptors and the actin network. This is how the speed of growth cone translocation usually reaches at most 1 µm/min, whereas the internal actin flow rate proceeds at a rate of several µm/min (Fig. 1A and B). Such slippage is best demonstrated by the use of optical tweezers to impose low forces on ligand-coated microspheres presented to the growth cone dorsal surface (Fig. 3A). Beads tend to move rearward as they couple to the actin flow, and then suddenly snap back into the trap center, when receptor-cytoskeleton bonds break¹⁶ (the force of optical tweezers is usually not enough to rupture ligand-receptor bonds, which remain intact at the cell surface). Thus, a step in which a nucleating cluster of adhesion receptors recruits a minimal number of intracellular partners allowing coupling to the actin flow, can be a rate-limiting factor in growth cone progression.Open in a separate windowFigure 2Molecular components involved in growth cone migration. (A) Top view diagram showing filopodia which sense the environment, a flat lamellipodium which is the site of actin dynamics and the thicker central domain and axon which contain dynamic microtubules. The plus signs are sites of actin polymerization and the minus signs indicate actin depolymerization. (B) Side view showing the life cycle of ligand/receptor adhesions.Open in a separate windowFigure 3Optical tweezers and FRAP experiments to measure ligand/receptor and receptor/cytoskeleton dynamics. (A) Optical tweezers experiments performed on ligand-coated beads placed on the growth cone dorsal surface.¹⁶ (B) The distance traveled rearward with respect to the trap center is measured. A 2 min trajectory is indicated in red. A pooled parameter called coupling index taking into account the latency for bead escape, as well as the mean velocity and lateral diffusion of the bead, measures the strength of receptor/cytoskeleton interactions. (C) FRAP experiments on membrane GFP-tagged molecules accumulated at ligand-coated microspheres having sedimented on growth cones. The fluorescence intensity is normalized to represent the receptor enrichment level at the bead contact. (D) The recovered intensity is fit by a diffusion/reaction model, which yields a collective equilibrium turnover rate of ligand/receptor bonds. In red is the average of a series of individual curves (grey).In contrast, when strong connection is formed and if the substrate is resistant enough, then the molecular clutch engages and the cell reacts. In growth cones from Aplysia bag cells, forces were imposed on microspheres coated with ApCAM (the homolog of vertebrate NCAM) using a microneedle to locally block the retrograde actin flow. This was systematically followed by a protrusion of the microtubule-rich central domain towards those stiff contacts and forward expansion of the actin-rich lamellipodium.⁹ These phenomena were later shown to be controlled by a src protein kinase.¹⁷ In the case of rat hippocampal neurons, a dramatic accumulation of actin at N-cadherin coated microspheres is observed when the latter are restrained from moving rearward by a microneedle.¹⁶ This phenomenon is mediated by a connection between N-cadherin and α-catenin, likely triggering local actin polymerization. By careful analysis of the bead trajectories at varying ligand densities and computation of the latency for bead escape when the optical trap is applied continuously, one can extract a quantitative index of receptor-cytoskeleton coupling (Fig. 3B). Overall, a strong correlation was observed between such coupling index and the velocity of growth cone migration on N-cadherin substrates, both by varying N-cadherin ligand density and by expressing mutated N-cadherin molecules, supporting the clutch concept.¹⁶ This mechanism is consistent with in vivo experiments showing that overexpression of the N-cadherin intracellular tail in retinal ganglion cells results in severely impaired axon outgrowth.¹⁸ As a negative example of the clutch model, beads coated with fibronectin (our unpublished data) or anti-α1 integrin antibodies³ couple weakly to the actin flow in growth cones while, in parallel, the migration of growth cones on fibronectin- or collagen-coated substrates is rather limited.^6,19Molecular mechanisms parallel to the “clutch” can also be involved in growth cone migration. For example, IgCAM adhesions can not only couple to the rearward actin flow but also to static components of the cytoskeleton. Indeed, a 30% fraction of TAG-1 or anti-L1 coated beads can stay immobile on the growth cone surface.^12,20 These contrasting behaviors are likely mediated by interactions between the IgCAM intracellular domain and different binding partners (ankyrin vs. ERM),¹³ and may be responsible for the pauses which alternate with phases of growth cone advance. Also, homophilic adhesions between molecules of cadherin-11 couple very weakly to the actin flow, but promote substantial growth cone migration when cadherin-11 is presented as a substrate. This effect seems to be mediated by an independent interaction with the FGF receptor, which triggers actin dynamic through a signaling cascade.^21,22As growth cones migrate, adhesion sites must be recycled at a rate that somehow matches the speed of migration. Adhesion turnover can be schematically decomposed in several sequential phases (Fig. 2B). (1) Initiation of a first single ligand/receptor bond powered by membrane diffusion²³ and followed by trapping through a key/lock interaction; (2) Formation of small adhesion clusters through the recruitment of more ligand/receptor pairs, and possibly stabilized by cis-oligomerization (cadherins through the same interface as the trans-dimer, IgCAMs through FnIII domains). These clusters might form very transiently and serve as sites of actin recruitment, as demonstrated for N-cadherin;¹⁶ (3) contact maturation and possible reinforcement by connection to the cytoskeleton (as demonstrated for integrins in fibroblasts²⁴); (4) Adhesion rupture, which can proceed through ligand/receptor dissociation triggered by cytoskeleton tension. Indeed, the intrinsic lifetime of ligand/receptor bonds such as cadherins, is sensitive to the mechanical force applied on them.²⁵ Furthermore, the loosening of receptor/cytoskeketon connections can cause inside-out rupture of ligand/receptor bonds. This was demonstrated for fibronectin/integrin interactions by the fact that when fibronectin coated-beads reach the base of a fibroblast lamellipodium, they spontaneously detach from the cell surface.²⁶ In the case of very sticky ligand/receptor interactions such as SynCAM homophilic adhesions,²⁷ this process can actually be a limiting step that slows down growth cone advance. Indeed, SynCAM couples very well to the actin flow, but is unable to support growth cone migration.¹⁶ Finally, adhesion rupture might also proceed through membrane rupture, the adhesion receptors being extracted from the cell membrane and left behind on the substrate (demonstrated for integrins at the tail of fibroblasts²⁸).These basic processes can be accompanied by more complex and active phenomena, e.g. involving forward surface transport as shown for NCAM²⁹ or internal trafficking in the case of L1.³⁰ By interacting with the clathrin adaptor AP-2 through a specific RSLE motif in its intracellular tail, L1 can undergo endocytosis in the central domain and exocytosis at the periphery of the growth cone.³⁰ This mechanism generates a density gradient of L1 molecules which accelerates the formation of bonds with a variety of ligands, including L1 itself. The use of an L1-GFP construct in which the N-terminal GFP could be rapidly cleaved off by thrombin, together with L1-Fc microspheres manipulated by optical tweezers showed that local exocytosis of L1-rich vesicles at the growth cone periphery indeed participates in enhancing the formation of L1 homophilic contacts.³¹ We did not observe such internal traffic for N-cadherin within the growth cone, partly because of a difficulty to introduce a fluorescent protein tag in the ectodomain, which otherwise perturbs the adhesive function. However, the use of an N-cadherin molecule with triple mutation in the juxta-membrane domain that abolishes binding to p120 catenin, involved in the export of N-cadherin to the cell surface, suggested that recycling events might also play a role.³²By measuring the fluorescence recovery after photobleaching (FRAP) of GFP-tagged receptors transiently trapped at ligand-coated microspheres and analyzing the curves using a diffusion/reaction model, we were able to compute the equilibrium turnover rates of ligand/receptor pairs in controlled adhesive contacts involving many simultaneous bonds (Fig. 3C and D). We found that mature L1 homophilic adhesions recycle fast compared to other IgCAMs such as TAG-1/NrCAM adhesions,²⁰ likely owing to the specific internalization motif present in L1. Indeed, the recycling rate was reduced by a factor of 3 after truncation of the L1 intracellular tail, which prevented endocytosis.³¹ N-cadherin homophilic adhesions have an intermediate turnover rate, which is sensitive to the binding to catenin partners.³² Using these measurements as well as data from the literature, we plotted the impact of both receptor-cytoskeleton coupling and adhesion turnover rate on neurite outgrowth (Fig. 4A and B), which is strongly proportional to growth cone velocity.¹⁶ The graphs show that a strong coupling between ligand-occupied receptors and the actin flow is necessary, but not sufficient for neurite extension (Fig. 4C). Another requirement is that the turnover of ligand/receptor adhesions lies in an optimal range: not too high, otherwise bonds detach before coupling can occur, and not too slow either, since sticky bonds which do not rupture paralyze growth cone progression (Fig. 4D). A similar bell-shape curve between the strength of cell-substrate adhesion and cell migration speed was demonstrated for fibroblasts³³ and keratocytes,³⁴ indicating that these coupled mechanisms are fundamental to cell migration. To fully understand the quantitative relationship between adhesion turnover and the clutch process, it would be helpful to add data to this preliminary graph. For example, the extracellular matrix molecule laminin is known to support axon growth very efficiently but, to our knowledge, neither the coupling to the actin flow in growth cones or the adhesive turnover rate of integrins has been evaluated yet. Conversely, NCAM was shown to couple well to the actin flow³⁵ and induce neurite outgrowth,^4,36 but measurements of the lifetime of NCAM homophilic adhesions within growth cones are still lacking.Open in a separate windowFigure 4Impact of ligand/receptor turnover rate and receptor/cytoskeletal coupling on neurite outgrowth. (A and B) Example of 2 DIV rat hippocampal neurons plated on N-cadherin-Fc coated substrate and transfected at 1 DIV with N-cadherin-GFP. (A) DIC image. (B) Fluorescence image. The longest neurite, most likely the axon, is outlined by arrowheads. (C and D) In both graphs, the y-axis represents the longest neurite length after two days plating on ligand-coated glass. (Red) Rat hippocampal neurons transfected with either wild type or mutated N-cadherin molecules, interacting with purified N-cadherin ligands.^16,32 The scale in red intensity represents from dark to light: wild type N-cadherin, N-cadherin deleted of the whole ectodomain, N-cadherin truncated in the C-terminal region binding to β-catenin, N-cadherin with triple mutation in the juxta-membrane domain interacting with p120, and wild type N-cadherin in the presence of cytochalasin D. (Blue) Neurons transfected with either wild type L1 (dark) or L1 truncated in the intracellular tail (light), interacting with purified L1.³¹ Neurite growth on L1 was estimated from references.^7,14,30 (Grey) Interaction between endogenous SynCAM1 molecules expressed on growth cones and SynCAM-Fc ligands coated on microspheres or flat glass.¹⁶ The turnover of SynCAM homophilic interactions was estimated from SynCAM-coated Quantum dots detaching from neurons transfected with SynCAM1.⁴² (Green) Neuroblastoma cells expressing NrCAM-GFP in contact with TAG-1 coated microspheres.²⁰ DRG neurite growth on TAG-1 was taken from reference.⁴³ (Orange) The coupling index was taken from optical tweezers experiments using anti-β1 integrin coated beads interacting with DRG neurons,³ the turnover rate was inferred from FRAP experiments on fibroblast focal contacts⁴⁴ and neurite growth on fibronectin was estimated from reference.¹⁹ We omitted statistics for clarity. The SEM are usually in the order of 5–15% of the mean, for sample sizes of typically 20–30 beads (coupling index and turnover rate) and 40–100 transfected cells (neurite length).One important question is how these observations obtained from simplified in vitro systems using stiff substrates of well-defined geometry coated with specific purified proteins at controlled density, translate to the in vivo situation. There, the 3D substrate is comprised of extracellular matrix and multiple cell types, co-expressing many different CAMs that can bind simultaneously in various stoechiometries and also generating local gradients of chemo-attractant and chemo-repulsive signals.³⁷ Substrate flexibility is probably an important factor, since axons grow more slowly when neurons are plated on a layer of fibroblasts expressing CAMs²¹ than when molecules are immobilized on a substrate.^16,30 This preference for cells to move on stiff substrates, called durotaxis, has been well described for fibroblasts.³⁸ Another specific feature of the in vivo situation is the existence of decision points, often correlated with the presence of guidepost cells where growth cones make a pause and often change shape and reorient before turning to another direction.³⁹ This type of behavior has been successfully mimicked in vitro using artificial guideposts made of fibronectin or laminin coated microspheres.⁴⁰ Whereas growth cones display a fairly continuous displacement on a homogeneous substrate,¹⁶ the presence of these guideposts make growth cones either slow down, pause or even collapse, or conversely accelerate, depending on the CAM grafted on the bead.^40,41 Finally, the shape itself of the growth cone can be an indicator of its motile state:³⁹ this is also true in vitro where small growth cones are often the most rapid whereas large and flat growth cones stay rather immobile. Thus, although the in vivo situation seems at first sight awfully complex, some general trends can be explained given a small number of interacting molecular species and rather simple bio-chemical and mechanical models.In conclusion, the dynamic regulation of growth cone advance can take place at several levels: (1) the actin-associated proteins controlling actin dynamics (nucleation, polymerization, sequestering, branching); (2) the activity of motors pulling on the actin network, generating the retrograde flow; (3) the intracellular adaptor proteins that link actin to the CAMs; (4) the membrane delivery and retrieval of CAMs; (5) the ligand/receptor interaction properties themselves; and (6) the processes regulating microtubule assembly and microtubule/actin interactions at the base of the growth cone. The orchestration in time and space of all these processes generates the movement and reactivity of growth cones necessary to lead axons to their target cells.     

Anchoring stem cells in the niche by cell adhesion molecules

Adult stem cells generally reside in supporting local micro environments or niches, and intimate stem cell and niche association is critical for their long-term maintenance and function. Recent studies in model organisms especially Drosophila have started to unveil the underlying mechanisms of stem anchorage in the niche at the molecular and cellular level. Two types of cell adhesion molecules are emerging as essential players: cadherin-mediated cell adhesion for keeping stem cells within stromal niches, whereas integrin-mediated cell adhesion for keeping stem cells within epidermal niches. Further understanding stem cell anchorage and release in coupling with environmental changes should provide further insights into homeostasis control in tissues that harbor stem cells.Key words: stem cell, niche, anchorage, cell adhesion, extracellular matrix, cadherin, integrinTissue-specific adult stem cells are characterized by their prolonged self-renewal ability and potentiality to differentiate into one or more types of mature cells. These unique properties make stem cells essential for maintaining tissue homeostasis throughout life. It is generally believed that all adult stem cells reside in specific microenvironments named niches, which provide physical support and produce critical signals to maintain stem cell identity and govern their behavior.^1–4 Consequently, intimate stem cell and niche association is a pre-requisite for stem cell''s long-term maintenance and function. How stem cells are kept within the niche is thus an important issue in stem cell biology. Characterization of a number of stem cell niches in model organisms has led to the classification of niches into two general types: stromal niches where stem cells have direct membrane contact with the niche cells and epidermal niches where stem cells are usually associated with the extracellular matrix (ECM), and do not directly contact any fixed stromal cells.¹ Studies in Drosophila have led to the cellular and functional verification of the stem cell niche theory^5,6 and not surprisingly, have also led to the discovery of the molecular mechanisms anchoring stem cells to the niche. Here I consider recent studies in Drosophila on types of cell adhesions used to anchor stem cells in the niches, and summarize cell adhesion molecules utilized in the most characterized niches in the mammalian tissues, and suggest that cadherin-mediated cell-to-cell adhesion and integrin-mediated cell-to-ECM adhesion are possibly two general mechanisms that function in respective stromal or epidermal niches for stem cell anchorage in diverse organisms.     

v-Crk regulates membrane dynamics and Rac activation

Cell migration is an integrated process that involves cell adhesion, protrusion and contraction. We recently used CAS (Crk-associated substrate, 130CAS)-deficient mouse embryo fibroblasts (MEFs) to examined contribution made to v-Crk to that process via its interaction with Rac1. v-Crk, the oncogene product of avian sarcoma virus CT10, directly affects membrane ruffle formation and is associated with Rac1 activation, even in the absence of CAS, a major substrate for Crk. In CAS-deficient MEFs, cell spreading and lamellipodium dynamics are delayed; moreover, Rac activation is significantly reduced and it is no longer targeted to the membrane. However, expression of v-Crk by CAS-deficient MEFs increased cell spreading and active lamellipodium protrusion and retraction. v-Crk expression appears to induce Rac1 activation and its targeting to the membrane, which directly affects membrane dynamics and, in turn, cell migration. It thus appears that v-Crk/Rac1 signaling contributes to the regulation of membrane dynamics and cell migration, and that v-Crk is an effector molecule for Rac1 activation that regulates cell motility.Key words: v-Crk, Rac, lamellipodia dynamics, cell migration, p130CASCell migration is a central event in a wide array of biological and pathological processes, including embryonic development, inflammatory responses, angiogenesis, tissue repair and regeneration, cancer invasion and metastasis, osteoporosis and immune responses.^1,2 Although the molecular basis of cell migration has been studied extensively, the underlying mechanisms are still not fully understood. It is known that cell migration is an integrated process that involves formation of cell adhesions and/or cell polarization, membrane protrusion in the direction of migration (e.g., filopodium formation and lamellipodium extension), cell body contraction and tail detachment.^1–3 Formation of cell adhesions, including focal adhesions, fibrillar adhesions and podosomes are the first step in cell migration. Cell adhesions are stabilized by attachment to the extracellular matrix (ECM) mediated by integrin transmembrane receptors, which are also linked to various cytoplasmic proteins and the actin cytoskeleton, which provide the mechanical force necessary for migration.^2,4 The next steps in the process of cell migration are filopodium formation and lamellipodium extension. These are accompanied by actin polymerization and microtubule dynamics, which also contribute to the control of cell adhesion and migration.⁵Focal adhesions are highly dynamic structures that form at sites of membrane contact with the ECM and involve the activities of several cellular proteins, including vinculin, focal adhesion kinase (FAK), Src family kinase, paxillin, CAS (Crk-associated substrate, p130CAS) and Crk.⁶ A deficiency in focal adhesion protein is associated with the severe defects in cell motility and results in embryonic death. For example, FAK deficiency disrupts mesoderm development in mice and delays cell migration in vitro,⁷ which reflects impaired assembly and disassembly the focal adhesions.⁸ In addition, mouse embryonic fibroblasts (MEFs) lacking Src kinase showed a reduced rate of cell spreading that resulted in embryonic death.⁹ Taken together, these findings strongly support the idea that cell adhesion complexes play crucial roles in cell migration.CAS is a hyperphosphorylated protein known to be a major component of focal adhesion complexes and to be involved in the transformation of cells expressing v-Src or v-Crk.¹⁰ CAS-deficient mouse embryos die in utero and show marked systematic congestion and growth retardation,⁴ while MEFs lacking CAS show severely impaired formation and bundling of actin stress fibers and delayed cell motility.^4,11,12 Conversely, transient expression of CAS in COS7 cells increases cell migration.¹¹ Crk-null mice also exhibit lethal defects in embryonic development,¹³ which is consistent with the fact that CAS is a major substrate for v-Crk, and both CAS and v-Crk are necessary for induction of cell migration.¹⁴ v-Crk consists of a viral gag sequence fused to cellular Crk sequences, which contain Src homology 2 (SH2) and SH3 domains but no kinase domain, and both CAS and paxillin bind to SH2 domains.^12,15,16 Despite the absence of a kinase domain, cell expressing v-Crk show upregulation of tyrosine phosphorylation of CAS, FAK and paxillin, which is consistent with v-Crk functioning as an adaptor protein.¹⁷ Moreover, this upregulation of tyrosine phosphorylation correlates well with the transforming activity of v-Crk.¹⁷ By contrast, tyrosine phosphorylation of FAK and CAS is diminished in Src kinase-deficient cells expressing v-Crk, and they are not targeted to the membrane, suggesting v-Crk signaling is Src kinase-dependent. After formation of the CAS/v-Crk complex, v-Crk likely transduces cellular signaling to Src kinase and FAK.¹² Notably, tyrosine phosphorylation of FAK and cell migration and spreading are all enhanced when v-Crk is introduced into CAS-deficient MEFs.¹² We therefore suggest that v-Crk activity, but not cellular Crk activity, during cell migration and spreading is CAS-independent.Membrane dynamics such as lamellipodium protrusion and membrane ruffling reportedly involve Rac1,¹⁸ α4β1 integrin,¹⁹ Arp2/3,⁶ and N-WASP,²⁰ and are enhanced in v-Crk-expressing CAS-deficient MEFs.²¹ Moreover, expression in those cells of N17Rac1, a dominant defective Rac1 mutant, abolished membrane dynamics at early times and delayed cell migration.²¹ v-Crk-expressing, CAS-deficient MEFs transfected with N17Rac1 did not begin spreading until one hour after being plated on fibronectin, and blocking Rac activity suppressed both membrane dynamics and cell migration. We therefore suggest that v-Crk is involved in cell attachment and spreading, and that this process is mediated by Rac1 activation. In addition, v-Crk expression apparently restores lamellipodium formation and ruffle retraction in CAS-deficient MEFs. Thus v-Crk appears to participate in a variety cellular signaling pathways leading to cell spreading, Rac1 activation, membrane ruffling and cell migration, even in the absence of CAS, its major substrate protein.In fibroblasts, the Rho family of small GTP-binding proteins (e.g., Cdc42, Rac and Rho) functions to control actin cytoskeleton turnover, including filopodium extension, lamellipodium formation and generation of actin stress fibers and focal adhesions.²² These GTPases function in a cascade, such that activation of Cdc42 leads to activation of Rac1, which in turn activates Rho.²² Once activated, Rho controls cell migration. Cell adhesion to ECM leads to the translocation of Rac1 and Cdc42 from the cytosol to the plasma membrane,²³ where they regulate actin polymerization at the leading edge.^19,24 Dominant negative Rac and Cdc42 mutants inhibit the signaling to cell spreading initiated by the interaction of integrin with ECM.²⁴ The fact that cellular levels of activated Rac are higher in cells adhering to ECM than in suspended cells further suggests that activation of Rac and Cdc42 is a critical step leading to membrane protrusion and ruffle formation. It is noteworthy in this regard that v-Crk is able to induce Rac activation and its translocation to plasma membrane.²¹Overall, the findings summarized in this article demonstrate that v-Crk participates in several steps leading to cell adhesion and spreading (Fig. 1), and the targeting of v-Crk to focal adhesion sites appears to be a prerequisite for regulation of cell migration and spreading via Rac activation. To fully understand its function, however, it will be necessary to clarify the role of v-Crk in Rac1 and Cdc42 activation initiated by integrin-ECM interactions.Open in a separate windowFigure 1Schematic diagram of v-Crk signaling in MEFs. Cell adhesion signaling initiated by the integrin-ECM interaction triggers v-Crk signaling mediated by Src kinase, after which focal adhesion proteins are tyrosine phosphorylated. These events lead to translocation of Rac from the cytosol to the membrane, where it promotes membrane protrusion and ruffle formation. Under basal conditions, Rac is bound with GDP and is inactive. Upon stimulation, Rac activation is mediated by guanine nucleotide exchange factors (GEFs) that stimulate the release of bound GDP and the binding of GTP. Activation of Rac is transient, however, as it is inactivated by GTPase activating protein (GAP).     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.