Myosin light chain kinases: Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.¹ A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.²One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.^1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.² Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.⁴ Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.⁵ Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.⁶ More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.^7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.^8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.^10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).¹¹ Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,¹¹ have been shown to colocalize on stress fibers.^12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.^14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.¹⁶ More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.¹⁷ The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.^9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.^17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.^17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.^20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.¹⁷ These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,^11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.¹¹Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).^23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.^8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.^23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.^26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,^26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),^18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,²⁹ MLCK only causes diphosphorylation when present at higher concentrations.³⁰ By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.^30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.^21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear²⁵ and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.²³ Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.¹⁷ These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.^11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.¹⁷The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.³⁴ GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,³⁵ and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

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     

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.     

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     

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

The process of epithelial lumenogenesis requires coordination of a network of signaling machinery communicated to each cell through subsequent cell divisions. Formation of a single hollow lumen has previously been shown to require Tuba, a Cdc42 GEF, for Cdc42 activation and correct spindle orientation. Using a Caco-2 model of lumenogenesis, we show that knockdown (KD) of the actin regulator N-WASP, causes a multilumen phenotype similar to Tuba KD. Defects in lumenogenesis in Tuba KD and N-WASP KD cells are observed at the two-cell stage with inappropriate marking of the pre-apical patch (PAP )—the precursor to lumen formation. Strikingly, both Tuba and N-WASP depend on each other for localization to the PAP. We conclude that N-WASP functions cooperatively with Tuba to facilitate lumenogenesis and this requires the polyproline region of N-WASP.Key words: lumen, N-WASP, tuba, E-cadherin, pre-apical patchMany epithelial tissues are organized as hollow tubes whose open lumina connect the body with its external environment.^1,2 These tubes consist of a monolayer of polarized cells that envelope the central lumen. Lumen formation is thus a key process in epithelial morphogenesis that depends upon cell polarity to establish three cell surface domains: a basal surface adherent to the extracellular matrix, a lateral surface between cells, and an apical surface that is exposed to the luminal fluids. Of note, the apical membrane is biochemically and morphologically distinct from the baso-lateral surfaces and effectively defines the luminal surface.^3,4For a lumen to form, cells must first mark the site at which apical membrane is to be inserted, something that is achieved at the first cell division.⁵ Targeted trafficking of apical membrane constituents defines a pre-apical patch (PAP), the precursor to the definitive lumen.⁵ Such insertion of apical membrane must presumably be coordinated with the assembly of apical junctions to segregate nascent apical from lateral membrane domains.² Subsequent cell divisions direct apical membrane and protein constituents to this point of initial apical membrane placement.⁶ Coordinated luminal positioning enables the initial formation of a single hollow lumen that subsequently expands through polarized fluid secretion to separate apical membranes, such as occurs in the embryonic gastrointestinal tract,⁷ or by apoptosis or autophagy of the central cells as is observed in mammary gland development.^8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina.^5,6Crucial to lumenal morphogenesis is then the mechanism(s) that mark the site where the PAP will form. Cdc42 signaling is increasingly implicated in this process,^2,10 with downstream consequences that include control of mitotic spindle orientation,⁵ which itself influences PAP placement⁵ and potentially regulation of cell-cell junctions. Like other Rho family GTPases, the subcellular location of Cdc42 signaling is determined by the action of upstream proteins, notably guanine nucleotide exchange factors (GEFs).^11,12 Of these, Tuba, a Cdc42-specific GEF,¹³ has emerged as a regulator of lumenal morphogenesis that controls PAP placement through mitotic spindle orientation.¹⁰Tuba is also a scaffolding protein¹³ capable of linking the actin assembly machinery with trafficking pathways. Not only is Tuba required for Cdc42 activation to direct spindle orientation,⁵ it also has the potential to interact with phosphoinositides that define the PAP.¹⁴ Additionally, Tuba binds directly to the actin regulator N-WASP, a key molecule in the organization of actin and itself a Cdc42 effector.¹⁵ Further, Tuba and N-WASP cooperate in various forms of actin-driven cellular motility, such as vesicle propulsion and cell invasive behavior.¹⁶ Interestingly, in epithelial cells N-WASP is also found at cadherin-based cell-cell junctions.¹⁷ In fact it has been proposed that N-WASP functions downstream of Tuba in the maintenance of epithelial junctional homeostasis as N-WASP overexpression was capable of rescuing a Tuba KD phenotype.¹⁸ Therefore, Tuba has the potential to play a central role in coordinating the molecular complexes required for productive polarization of epithelial cells and placement of the PAP during lumenogenesis. However, whether other protein interactions contribute to the morphogenetic impact of Tuba remain to be assessed.Three-dimensional cell culture systems are being utilized to identify critical components in lumen formation. In particular, Madin-Darby canine kidney cells (MDCK) and Caco-2 gastrointestinal cells are commonly used to study cyst and/or tubule formation. MDCK cells undergo both cyst and tubule growth, apoptosis being primarily responsible for the final step in lumen formation,¹⁹ while Caco-2 cells primarily utilize fluid influx to expand cysts.⁵ Cyst culture systems replicate aspects of in vivo organogenesis²⁰ providing tangible, powerful models to analyze and dissect the coordinated cellular mechanisms and processes that occur during epithelial morphogenesis.In this study we examined the relationship between Tuba and N-WASP in early epithelial lumenogenesis using Caco-2 three dimensional cyst cultures. Both Tuba and N-WASP RNAi cell lines result in mature cysts with multiple lumina, and at the two-cell stage, formed multiple PAPs. Interestingly, N-WASP KD perturbed Tuba localization at the PAP, however, N-WASP localization to the PAP was not affected to the same extent by Tuba KD. Taken together, these results suggest a complex interrelationship between Tuba and N-WASP for the coordinated formation of a single hollow lumen.     

The dual role of the ligand UNC-6/Netrin in both axon guidance and synaptogenesis in C. elegans

The extracellular cue UNC-6/Netrin is a well-known axon guidance molecule and recently it has also been shown to be involved with localization of pre-synaptic complexes. Working through the UNC-40/DCC/Fra receptor, UNC-6/Netrin promotes the formation of pre-synaptic terminals between the pre-synaptic AIY interneuron and its post-synaptic partner, the RIA interneuron. In the DA9 motor neuron, UNC-6/Netrin has an alternate role promoting the exclusion of pre-synaptic components from the dendrite via its UNC-5-receptor. Surprisingly, the requirement for UNC-5 persists even after DA9 axon migration is complete, because synapses become mis-localized after it is depleted. This observation provides at least a partial explanation for the persistence of UNC-6/Netrin and UNC-5 in the adult nervous system. These activities parallel the previously known bi-functional axon guidance effects of UNC-6/Netrin, since it can attract cells and axons expressing UNC-40/DCC/Fra and repel those expressing UNC-5 alone or in combination with UNC-40. UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from compartments within the DA9 axon, so that they only occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Regulation of both axon guidance and synapse formation by axon guidance cues permits coordination in circuit assembly between pre- and post-synaptic cells.Key words: nervous system development, axon guidance, synaptogenesis, Netrin/UNC-6, UNC-40/DCC/Fra, UNC-5, LIN-44/Wnt, EGL-20/Wnt, LIN-17/FrizzledDuring development of the nervous system, differentiated pro-neural cells become polarized and send out processes from the cell body that later become dendrites and axons. The pro-neural cells themselves and later their axons, often migrate long distances to their eventual targets using guidance cues.¹ Once the destination is reached, the axon usually selects among several available targets and establishes synapses with the correct post-synaptic partner. The synapse is the site of communication between the pre- and post-synaptic cell and many of the molecules involved in synapse formation are known.² Development of both the pre- and post-synaptic cells needs to be orchestrated to ensure that they are available to form synapses with each other and this process can be directed by guidepost cells. In organisms such as vertebrates, the guidepost cells are often glia, which guide two neurons to ensure that the correct synapse is formed.³ A case is presented here where glial cells secrete a cue to control the localization of pre-synaptic complexes in C. elegans. One notable aspect of this process is that the glial-secreted cue is a well-known axon guidance molecule, namely Netrin/UNC-6, but here it plays an additional and surprising role in selecting the site for the construction of a pre-synaptic complex.⁴UNC-6/Netrin, is a well-known bi-functional axon guidance cue that can attract some axons and repel others. It is a laminin-related molecule, originally isolated from C. elegans, with homologues in higher organisms.^5–7 UNC-6 has two receptors in C. elegans: UNC-40 and UNC-5.^8,9 Both have homologues in higher organisms: UNC-40/DCC/Fra (Deleted in Colorectal Cancer in vertebrates/Frazzled in Drosophila) and UNC-5/Unc5.^6,7,10 UNC-6/Netrin is expressed by cells mostly located in the ventral regions of C. elegans where it attracts many cells and axons expressing the receptor UNC-40.^8,11 Conversely, UNC-6/Netrin repulses axons and cells expressing UNC-5 alone, or in combination with UNC-40.^9,12 One aspect of this developmental process, however, that has been somewhat puzzling has been the observation that expression of both UNC-6 and UNC-5 persist into adulthood.^11,13 A partial explanation for the persistence of UNC-6 and UNC-5 is provided by Poon et al.¹⁴ who found that UNC-5 is required for both the initial polarized localization and maintenance of the pre-synaptic complexes in the DA9 motor neuron axon in C. elegans. The mechanisms used by the proteins in these new roles have not been established, but the localization of both UNC-5 and UNC-40 in the axons is controlled by their normal ligand, UNC-6/Netrin.UNC-40/DCC/Fra plays two independent roles in establishing the connection between the pre-synaptic AIY amphid inter-neuron and its post-synaptic partner, the RIA inter-neuron in the nerve ring of C. elegans, since it is involved in both axon guidance of RIA and synapse localization in AIY.⁴ The two neurons are located in the head region, close to the nerve ring and can be visualized using cell-specific markers (Fig. 1). UNC-6/Netrin plays a conventional guidance role in directing migration of the post-synaptic inter-neuron RIA, since the ventral trajectory of its axon is altered in the absence of UNC-40. The axon of the AIY inter-neuron migrates anteriorly from its cell body, then dorsally and synapses onto three other interneurons: RIA, AIZ and RIB. The AIY axon usually migrates normally without the UNC-40 receptor, which is not surprising as it does not make a ventral migration.Open in a separate windowFigure 1A schematic of the region close to the head of C. elegans is shown where the synapses between the pre-synaptic AIY interneuron (red) and the post-synaptic RIA interneuron (blue) occur. The pre-synaptic regions are shown as black dots. The glial cell (sheath cell) that is the source of UNC-6/Netrin is shown in green. The insert below the schematic shows the regions of the AIY axon divided into zones 1, 2 and 3 that are discussed in the text. Redrawn from Colon-Ramos et al.⁴ and WormAtlas²⁴ (with permission).Pre-synaptic complexes in AIY were detected by expression of fluorescently-tagged synaptic vesicle associated RAB-3.⁴ They were found mainly in the “elbow” region (Fig. 1, zone 2) and about eight more complex-containing areas were found in the region most distant from the cell body within the nerve ring in wild-type animals (Fig. 1, zone 3). A hypomorphic allele of unc-40, wy81, was found in a genetic screen for mutants exhibiting altered localization of pre-synaptic complexes. In the absence of fully functional UNC-40, the pre-synaptic markers were not observed in zone 2, but were present in the more distal region, zone 3. In addition, the pre-synaptic region (zone 2) of AIY did not have an expanded diameter in the manner characteristic of this particular synapse, as detected by electron microscopy. The synapses between AIY and RIA in the absence of UNC-40 were abnormal in several other respects. There was a severe reduction in the active zone proteins ELKS-1/ERC/CAST and SYD-2/α Liprin, suggesting a defect in the pre-synaptic differentiation of AIY. Pre-synaptic defects in AIY caused by absence of UNC-40 could only be rescued by cell-autonomous expression of the receptor.Localization of UNC-40/DCC/Fra in the AIY interneuron is controlled by UNC-6/Netrin emanating from a pair of glial cells called the ventral cephalic sheath cells (VCSCs), which are similar to astrocytes.⁴ Wadsworth et al.¹¹ have previously shown that the VCSCs at the nerve ring express UNC-6/Netrin during neurulation. Colon-Ramos et al.⁴ found the VCSCs project deeply invaginated end-feet that form membranous lamellae, thereby ensheathing the region of AIY-RIA synapses. There is thus a very tight association between the glial cell and the two interneurons in the region of the synapses in zone 2. UNC-40 localizes to the pre-synaptic zones 2 and 3. In the absence of UNC-6, UNC-40 is more diffuse and is present along the entire neuron.The anatomical relationship between the sheath cells and synapses is instructive in mediating AIY:RIA innervations. Sheath cell morphology was altered by the absence of UNC-34/Enabled such that the glial end-feet now migrated further posteriorly to include zone 1.⁴ There was a concomitant appearance of both ectopic pre-synaptic complexes and UNC-40 localization in zone 1 due to an alteration in the source of UNC-6. In UNC-34/Enabled mutants, the trajectory of the RIA interneuron was also altered, such that it had migrated towards the new site of the synapses. Therefore, in this study UNC-40 is playing two independent roles, one in axon path-finding of the RIA axon and a second in positioning the synapses in the AIY pre-synaptic cell. Both of these activities are under the control of UNC-40''s normal ligand, UNC-6/Netrin, that is expressed by glial cells that ensheath the region of the synapses. Regulation of both processes by a single molecule allows co-ordination in circuit assembly.In contrast to the work described above, UNC-6/Netrin and its receptor UNC-5 have recently been reported to exclude synaptic vesicle and active zone components from the dendrite of the DA9 motor neuron in C. elegans (Fig. 2).¹⁴ The DA9 neuron can be divided into five zones (see insert in Fig. 2). It synapses en passant with the VD/DD motor neurons and the body wall muscles along the dorsal cord. In wild-type animals, the synapses of the DA9 neuron were detected using a fluorescently labelled RAB-3, a synaptic vesicle associated protein, and they were found mainly in the region most distant from the cell body (zone 5 of DA9 in Fig. 2). Synapses were entirely excluded from the dendrite (zone 1) and the remainder of the axon (zones 2, 3 and 4) in wild-type animals. Interestingly, UNC-5 had a somewhat complementary distribution to the pre-synaptic complexes since it was expressed mainly in the dendrite of DA9 and the ventral region of the axon (zones 1 and 2 in Fig. 2), suggesting that the presence of UNC-5 can exclude synapses.Open in a separate windowFigure 2The tail region of C. elegans is shown with the DA9 motor neuron. The DA9 axon is in blue and the dendrite in orange. The pre-synaptic structures are in black. The sources of the LIN-44/Wnt, EGL-20/Wnt and UNC-6/Netrin are shown. The insert below shows the various zones of the DA9 neuron described in the text. Redrawn from Poon et al.¹⁴ and WormAtlas²⁴ (with permission).The correct location of the pre-synaptic complexes in DA9 is dependent on both the ligand UNC-6/Netrin and the receptor UNC-5. RAB-3 was found ectopically in the dendrites of either unc-6(ev400), or unc-5(e53), both considered to be null mutants.^5,9,15 Other pre-synaptic vesicle proteins tested, including SNB-1/Synaptobrevin and SNG-1/Synaptogyrin, as well as CCB-1, an L-type voltage-gated calcium channel β subunit and the active zone protein SYD-2/α-liprin, were also mis-localized in the dendrite in the absence of either UNC-6 or UNC-5.¹⁴ UNC-5 functions cell autonomously for the exclusion of pre-synaptic complexes. Interestingly, deletion of either one of the immunoglobulin domains or one of the thrombospondin domains from the extracellular regions of an UNC-5 protein was previously shown to alter the sub-cellular localization of the protein so that is was more localized to the cell body than wild-type UNC-5.¹⁵ This suggests that the extracellular region of UNC-5 is responsible for its localization in the neuron and it would be interesting to see if synapse localization is affected in the absence of the extracellular domains.In addition to its roles in axon guidance and localizing pre-synaptic complexes, an ongoing supply of UNC-5 is required in DA9 to maintain the position of the synapses. This has been demonstrated by the use of a temperature-sensitive silencing intron construct that allowed UNC-5 expression at a permissive temperature of 25°C but not at the restrictive temperature of 16°C.¹⁴ Temperature shift experiments from the permissive temperature to the restrictive temperature at the L4 stage, after the axon was already fully developed, caused synapse mis-localization similar to that observed in the absence of UNC-5. Initial synapse mislocalization was irreversible as the reverse shift from the restrictive to the permissive temperature at L4 failed to rescue the defect. The exclusion of pre-synaptic complexes from all the compartments of DA9 except for the most distal regions (zones 4 and 5) was not simply a consequence of axon misguidance, since axons that were not misguided due to the absence of UNC-5, still exhibited altered RAB-3 localization. Additionally, animals lacking another axon guidance cue, UNC-129/TGFβ exhibited misguidance of DA9 but not mis-localization of pre-synaptic components. Dendritic localization of the pre-synaptic proteins was also not just a reversal of the axons and dendrites in DA9, since four different dendritic proteins were correctly localized in the absence of both UNC-6/Netrin and UNC-5. The need for an ongoing supply of UNC-5 accounts for the observation that both UNC-5 and UNC-6 persist into adulthood, long after axon guidance or synapse formation in worms.^11,13 The finding that UNC-5 must be present on an ongoing basis to maintain localization of pre-synaptic complexes suggests a novel role for UNC-5 in maintaining the polarized localization of the pre-synaptic complexes in a manner independent of axon guidance or initial synaptic polarization. This is an intriguing finding and one that deserves investigation for other neurons and axon guidance molecules.Two Wnt cues also control synapse localization in the DA9 neuron but in different regions than UNC-6/Netrin.¹⁶ LIN-44/Wnt emanating from the tail region (light pink patch in Fig. 2) causes exclusion of synapses from the more posterior section of the DA9 axon located in the dorsal cord (zone 4 in Fig. 2). A second Wnt, EGL-20 is also produced by tail cells (deeper pink region in Fig. 2), and it excludes synapses from the region of the axon in the ventral cord (zone 2 in Fig. 2). Both Wnts cooperate to exclude synapses from zone 3. There is a strict correlation between the presence of the LIN-44/Wnt receptor, LIN-17/Fz, in zones 2, 3 and 4 and the absence of synapses in these regions. LIN-17/Fz is required cell-autonomously in DA9 to rescue synaptic localization defects. In the absence of LIN-44/Wnt, both the receptor LIN-17/Fz and the pre-synaptic complexes were mis-localized since they were now found in both zone 4 and 5 of the axon. Therefore, LIN-44/Wnt is instructive in regulating the location of the synapses in the axon of the DA9 neuron. Both LIN-44/Wnt and EGL-20/Wnt normally work cooperatively to exclude synapses, since animals lacking both had synapses in zones 3, 4 and 5 of DA9.UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from particular regions of the DA9 axon and only allow them to occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Ectopic expression of UNC-6/Netrin and LIN-44/Wnt in various cells and genetic backgrounds was used to show that UNC-6/Netrin and LIN-44/Wnt could function interchangeably in excluding synapses in the DA9 neuron.¹⁴ Ectopic expression of UNC-6 in a posterior to anterior gradient close to DA9 caused RAB-3 to be localized more posteriorly in zone 5, rather than in both zone 4 and 5. The mis-localization was UNC-5 dependent and was seen regardless of whether or not DA9 was misguided. Ectopic UNC-6 could also rescue mis-localization defects in the absence of either LIN-44/Wnt or its receptor LIN-17/Frizzled. These observations suggest that UNC-6/Netrin and LIN-44/Wnt both exclude synapses and can function together to control both axon guidance and pre-synaptic complex localization. Therefore, EGL-20/Wnt and LIN-44/Wnt work cooperatively with the UNC-6/Netrin ligand to inhibit the assembly of pre-synaptic complexes from inappropriate neuronal compartments. Synapses are excluded from the dendrite (zone 1) by UNC-6/Netrin, the region of the axon proximal to the cell body (zone 2) by EGL-20/Wnt, the commissures (zone 3) by EGL-44/Wnt and EGL-20/Wnt, and the distal portion of the axon (zone 4) by LIN-44/Wnt.¹⁴It remains to be seen whether UNC-6/Netrin and its receptors are usually involved in synapse localization in C. elegans itself and in other organisms, beyond the highly specific cell contexts discussed. The involvement of these molecules in both axon guidance and synaptogenesis is likely to be a general phenomenon, as the Netrins are expressed in the adult nervous systems of vertebrates including neurons and oligodendrocytes in the adult rat.¹⁷ DCC is expressed in the adult rat forebrain.¹⁸ UNC-5 is expressed in the heart and brain of adult vertebrates.¹⁹ Ephrins have also been shown to be involved in both axon guidance and synapse formation.²⁰ Wnts have been found to play roles in regulating neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation in invertebrates and mammals.²¹ Recently, it was shown that pro- and anti-synaptogenic effects of Wnt proteins are associated with the activation of canonical and non-canonical Wnt signaling pathways in Drosophila and mouse.^22,23 It is anticipated that many more instances of axon guidance molecules involved in synapse formation will be described. For instance, in the case of the synapse between the AIY and the RIA interneurons just discussed, AIY also synapses with two other interneurons, the AIZ and RIB but these synapses are not altered significantly in the absence of UNC-40/DCC/Fra. Presumably, these synapses require other molecules to guide synapse formation. Although the two receptors UNC-40 and UNC-5 are functioning with their normal ligand UNC-6/Netrin, it is not clear whether the remainder of the signaling pathways are conserved, and this question will be an interesting topic for future work on synapse formation.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle.As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90⁺ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.Key words: CD90, mesenchymal stem cells, hair follicle, dog.The hair follicle represents an important stem cell niche in the skin. It contains dermal and epithelial stem populations that display distinct properties and localization. While epithelial stem cells reside in the middle region of the hair follicle outer root sheath (Schneider et al., 2009; Lyle et al., 1998; Cotsarelis et al., 1990), dermal stem cells are located in the dermal sheath (DS) (Jahoda, 2003; Jahoda and Reynolds, 2001).The dermal sheath, or fibrous root sheath, is a layer of dense connective tissue that extends along the hair follicle, from the bulb up to the infundibulum. In the anagen hair follicle, it is comprised of mesenchymal cells located among collagen and elastic fibres.The cells are flat and elongated while collagen fibres form a circular inner layer and a longitudinal outer layer in the lower part of hair follicle (VonTscharner and Suter, 1994; Jahoda et al., 1992). At the base of the hair follicle, the DS is connected to the dermal papilla (Scott et al., 2000). The basement membrane, or glassy membrane, separates the DS from the epithelial component of the hair follicle (Scott et al., 2000).Follicular dermal stem cells have a mesenchymal origin and share many properties common to bone marrow-derived mesenchymal stem cells (MSCs) (Hoogduijn et al., 2006). They express the MSC cell-surface marker CD90, show a high colony forming unit ability and can differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes and myocytes (Hoogduijn et al., 2006; Jahoda et al., 2003). They also express neuroprogenitor markers (Hoogduijn et al., 2006) and, finally, they can repopulate the haematopoietic system (Lako et al., 2002). In the literature, we can find different information about stem cell localization: the whole dermal sheath, the peri-bulbar dermal sheath, the dermal papilla (Hoogduijn et al., 2006, McElwee et al., 2003, Gharzi et al., 2003, Jahoda et al., 2003.)CD90 (Thy-1) is a small GPI-anchored protein localized in the outer leaflet of the cell membrane (Low and Kincade, 1985). This protein is present in a large number of tissues and cells, even if a great species variation has been described (Mansour Haeryfar, 2004; Tokugawa et al., 1997; McKenzle and Fabre, 1981). CD90 plays a role in cell-cell interaction events, including intracellular adhesion and cell recognition during development (Saalbach et al., 2000; Morris, 1985), and is considered an important stem cell marker; for this last reason it is commonly used to identify mesenchymal stem cells in vitro (Kern et al., 2007; Yoshimura et al., 2006; Le Blanc and Ringdén, 2006; Pittenger et al., 1999). Furthermore, it has been identified in other kinds of stem cells such as haematopoietic progenitor cells (Craig et al., 1993) and hepatic progenitor cells in the human fetal liver (Masson et al., 2006).The hair follicle is the focus of increasing interest because it contains well defined stem cell populations that exhibit various developmental properties. We retain that in dogs, as already demonstrated in other species (Hoogduijn et al., 2006; Zhang et al., 2006; Jahoda et al., 2003; Lako et al., 2002), this organ may be a suitable and accessible source for both epithelial and mesenchymal stem cells that may be isolated and in vitro cultured. Since it is possible to take skin samples without injuring the patient, we chose the hair follicle to study and identify stem cells with the future purpose of using them in regenerative medicine.Dogs are affected by several skin diseases and some of them may be related to alterations of somatic stem cells. We retain that the study of hair follicle stem cell biology may improve our knowledge of etiology and pathogenesis of these skin diseases.In previous works we investigated the stem cells in dog hair follicles; we identified the location of putative epithelial stem cells at the isthmus and described the bulge-like region (Pascucci et al., 2006; Mercati et al., 2008). To the authors’ knowledge, there are no data available neither concerning the localization of DS stem cells nor concerning the expression of CD90 in the hair follicle as regards the canine species. Therefore, in this study, we described the morphological characteristics of DS cells and examined the immunohistochemical localization of CD90 protein in dog hair follicles with both light and transmission electron microscopy. The aim of our study is to observe the dermal sheath cells encompassing the hair follicle and to determine where CD90⁺ cells reside. CD90 is one of the main markers used to identify mesenchymal stem cells and it has been observed in stem cells isolated from the dermal sheath of hair follicles (Hoogduijn et al.,2006). For this reason, we suppose that CD90 protein can help us to identify the hair follicle dermal stem compartment in dog.