The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs

Background

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs).

Methods

Deep partial thickness burn model in Sprague–Dawley rats and the heat denatured cell model (52 °C, 35 s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin.

Results

Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs.

Conclusions

The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF.

General significance

We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis.     

Collective cell migration of smooth muscle and endothelial cells: impact of injury versus non-injury stimuli

Background

Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration – some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces.

Results

Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour.

Conclusion

For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

     

Early molecular events during the interaction of enveloped riboviruses with cells

A kinetic model was constructed and partly solved to describe the migration of the fluorescence label 1,6-diphenylhexatriene (DPH) in both directions when enveloped viruses, labelled with DPH in their envelopes are in contact with unlabelled cells or cell labelled in their membranes are in contact with unlabelled enveloped viruses. The central assumption is that two types of receptor sites exist on the cell surface, i.e., physical adsorption sites (P-sites), available to all the viruses studied in these papers and binding sites (B-sites) available only to the viruses which penetrate into the specific cells.The differential equations for the label migration, for different values of the ratio number of viruses number of sites were numerically solved, assuming different fractions of P- and B-sites.The equations also describe, appropriately the mechanism of rapid label migration in the system and substantiate the magnitude time of residence of the nonpenetrating viruses adsorbed on the cell surface. The resulting curves match satisfactorily those for the label release by the viruses and account well for the steady state values of the kinetics of label migration in the virus-cell system.     

Contact guidance of Walker carcinosarcoma cells by the underlying normal fibroblasts is inhibited by RGD-containing synthetic peptides

The metastatic spread of malignant neoplasms is associated with active migration of cancer cells. The migration of neoplastic cells during the metastatic process may be affected by various extracellular factors, including chemoattractants, haptotactic signals, electric fields, substrate anisotropy, and cell-to-cell contacts. We examined the effect of homotypic collisions and heterotypic interactions with normal human skin fibroblasts on the motile activity of Walker carcinosarcoma cells. It was found that Walker carcinosarcoma cells moving in a dense population neither show contact inhibition of movement when colliding with one another nor increase their motile activity as a result of contact stimulation of motility. On the other hand, when plated onto the surface of aligned fibroblasts, Walker carcinosarcoma cells migrated mainly along the long axes of underlying fibroblasts as a result of contact guidance. The directional character of movement (but not the speed of migration) of Walker carcinosarcoma cells on the surface of aligned fibroblasts was completely effaced by RGD-containing synthetic peptide at a concentration of 1 mg/ml but not by 5 microM verapamil (selective voltage-gated calcium channel inhibitor) or 10 microM gadolinium chloride (non-specific blocker of mechanosensitive ion channels). The suppression of directional character of migration of tumour cells by RGD-containing peptide was associated with the decrease in the amount of fibronectin macromolecules attached to fibroblasts. This suggests that alignment and anisotropic distribution of fibronectin macromolecules may be responsible for contact guidance of tumour cells moving on the surface of fibroblasts.     

Prioritising guidance cues: directional migration induced by substratum contours and electrical gradients is controlled by a rho/cdc42 switch

Coordinated cell migration is a fundamental feature of embryogenesis but the intracellular mechanism by which cells integrate co-existing extracellular cues to yield appropriate vectoral migration is unknown. Cells in the cornea are guided by a naturally occurring DC electric field (EF) (electrotaxis) as they navigate non-planar substrata but the relative potencies of electrotaxis and guidance by substratum shape (contact guidance) have never been determined. We tested the hypothesis that vectoral migration was controlled by selective activation of rac, cdc42 or rho in response to a 150 mV/mm EF or to a series of parallel substratum nanogrooves (NGs) 130 nm deep. EFs and NGs were presented singly or in combination. Electrotaxis of dissociated bovine corneal epithelial cells (CECs) on planar quartz required signalling by cdc42 and rho but not rac. Contact guidance by substratum NGs required rho but not cdc42 or rac activities. When an EF and NGs were superimposed in parallel, cathodal electrotaxis along NGs was enhanced compared to that on planar quartz but when they were superimposed orthogonally (vertical NGs with horizontal EF) cells were recruited from contact guidance to electrotaxis, suggesting that the EF was more potent. However, increasing the EF to 250 mV/mm was insufficient to recruit the majority to electrotaxis. Consistent for the cues in isolation, when an EF (150 mV/mm) and NGs were superimposed orthogonally, rac activity was not essential for either contact guidance or electrotaxis. However, attenuation of cdc42 signalling abolished electrotaxis and enhanced contact guidance relative to controls (no drug), whereas inhibiting rho signalling enhanced electrotaxis and rho stimulation enhanced contact guidance. Our data are consistent with the idea that migrating CECs use a cdc42/rho “switch” to sort vectoral cues, with cdc42 controlling electrotaxis and rho controlling contact guidance.     

Paradoxical perpendicular contact guidance displayed by mouse cerebellar granule cell neurons in vitro

In order to study migration of neurons in vitro, we cultured microexplants of the newborn mouse cerebellum outer layer, which is rich in immature granule cells, on a substratum double-coated with poly-L-lysine and laminin. The granule cells first migrated away from the explant along radially oriented parallel bundles of their neurites, thus displaying typical contact guidance. Then, in almost all explants, they changed their orientation by 90 degrees to extend cell processes and translocate perpendicular to the radial neurites. Orientation and migration of neurons perpendicular to the aligned parallel structure is a novel type of contact-guided cell behavior, and may have interesting implications in migration of neurons in the cerebellum and other parts of the nervous system.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.     

Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers and (4) stop signals locally expressed in the outmost layer of cortex.Key words: radial migration, cortex, guidance, semaphorin, diffusible factors, growth coneThe mammalian cerebral cortex has the typical laminar structure, the formation of which is essential for neurons in each cortical layer to establish the specific input and output connections with other brain regions. The development of the cortical laminar structure is known to involve the well-coordinated radial migration of newborn pyramidal neurons during development.¹ After young neurons are generated from the ventricular zone (VZ) and subventricular zone (SVZ), they leave their birthplace and migrate along radial glial filaments toward the surface of cortical plate (CP), crossing existing cortical layers composed of earlier born neurons and eventually settling down beneath the marginal zone (MZ, layer I).^1–3 It is generally accepted that the adhesion between neurons and radial glial filaments provides the directionality for these young neurons, and the targeting of neurons to specific lamina was controlled by the selective detachment of migrating neurons from radial glial fibers upon reaching the designated cortical layer.^2,3 However, we believe that the radial glial fibers can only serve as the adhesive scaffold for migrating neurons and constrain their migration in the radial dimension; it remains an open question regarding the nature of the signals that cause newborn neurons to migrate consistently outward along the fiber rather than inward. Whether the radial migration of cortical neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the VZ is entirely unknown, a potential guidance mechanism that has long been overlooked.Recently we found that the radial migration of layer II/III cortical neurons during development is guided by an extracellular guidance molecule semaphorin-3A (Sema3A).⁴ We observed that Sema3A is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is expressed at a high level in migrating neurons. By in utero electroporation, we were able to monitor the migration of a subpopulation of cortical neurons in their native environment and examine the effect of perturbing Sema3A signaling. We found that downregulation or conditional knockout of NP1 in young neurons impeded their radial migration with severe misorientation of affected neurons during their migration without altering their cell fate. Studies in cultured cortical slices further showed the requirement of the endogenous gradient of Sema3A for the proper migration of newborn neurons. Results from transwell chemotaxis assays in dissociated culture of newborn cortical neurons also supported the notion that Sema3A attracts the migration of these neurons through the receptor NP1. Thus, Sema3A may serve as a chemoattractive guidance signal for the radial migration of newborn cortical neurons toward upper layers. This is the first demonstration that radial migration of cortical neurons is guided by gradient of extracellular guidance factors. This study also suggests that guidance factors may guide the radial migration by their actions on the growth cone of the leading process of migrating neurons, via mechanisms similar to that found for their actions on axon guidance and dendritic orientation, followed by long-range cytoplasmic signaling that coordinates the forward motility of the entire neuron.⁵In this study, we have only observed an attractive effect of Sema3A in the radial migration of the layer II/III cortical neurons. However, to form the highly ordered laminar structure of the cortex, the entire process of neuronal migration is likely to depend on coordinated actions of multiple factors in the developing cortex, including other semaphorin family members and other guidance molecules, e.g., slits⁶ and ephrins,⁷ which are also expressed in the CP. We hypothesize that four groups of extracellular factors orchestrate to promote the proper radial migration and cortical lamination: (1) factors that are expressed in superficial layers of cortex and in a descending gradient, like Sema3A, may attract the upward migration of newborn neurons (attractive factors), (2) factors enriched in the VZ may exert repulsive action and help to “push” newborn neurons out of their birthplace (repulsive factors), (3) those factors widely expressed in all cortical layers may promote the motility of migrating neurons (pro-migratory factors) and (4) Some repulsive cues may be locally expressed in the superficial layer of cortex to prevent the over migration of neurons when they have arrived at the outmost layer (stop signal). Under the guidance of these four groups of factors, newborn neurons migrate all the way from VZ to the outmost layer of CP and then settle down. One of our recent tasks is to try to identify these four groups of factors.If the radial migration and cortical lamination are guided by diffusible factors, why is radial glial system necessary for this migration process? In other words, why earlier-born neurons in different layers cannot provide the supportive adhesion to young neurons during their radial migration? A potential explanation is that neurons in cortex undergo maturation after terminating their migration, accompanying with changes in their expression profiles of adhesion ligands, and become less and less supportive to the neuronal migration. In contrast, as a kind of cortical progenitor cells, radial glial cells maintain a relatively ‘young’ state and continue to express supportive adhesion ligands over a very long developmental stage. Thus, only the radial glial filament is capable of providing a bridge for newborn neurons to migrate over a very long distance across the non-permissive cell layers. In summary, we believe that during the cortical radial migration, signals from diffusible factors override the adhesive signal from radial glial fibers to promote the appropriate migration and placement of newborn neurons.? Open in a separate windowFigure 1A schematic diagram for the guidance of cortical radial migration by diffusible factors. (A) A model for the distribution of four groups of guidance factors in developing cortex. Radial glial filaments are shown in red, young neurons are in green. There may exist a descending gradient of attractive factors in upper cortical layers (yellow) and an ascending gradient of repulsive factors (blue) near the ventricular zone (VZ). Stop signals (purple) may come from the surface of cortex, and pro-migratory factors (dots) may be widely distributed. (B) Representative image of EGFP-labeled neurons migrating along radial glial filaments in the cortical tissue of E20 mouse. Sections were counterstained with DAPI (Red). Scale bar, 100 µm.     

NADPH Oxidase 1 Controls the Persistence of Directed Cell Migration by a Rho-Dependent Switch of α2/α3 Integrins

NADPH oxidase 1 (Nox1) is expressed mainly in colon epithelial cells and produces superoxide ions as a primary function. We showed that Nox1 knockdown inhibits directional persistence of migration on collagen I. This paper dissects the mechanism by which Nox1 affects the direction of colonic epithelial cell migration in a two-dimensional model. Transient activation of Nox1 during cell spreading on collagen 1 temporarily inactivated RhoA and led to efficient exportation of α2β1 integrin to the cell surface, which supported persistent directed migration. Nox1 knockdown led to a loss of directional migration which takes place through a RhoA-dependent α2/α3 integrin switch. Transient RhoA overactivation upon Nox1 inhibition led to transient cytoskeletal reorganization and increased cell-matrix contact associated with a stable increase in α3 integrin cell surface expression. Blocking of α3 integrin completely reversed the loss of directional persistence of migration. In this model, Nox1 would represent a switch between random and directional migration through RhoA-dependent integrin cell surface expression modulation.The two well-recognized defining hallmarks of cancer are uncontrolled proliferation and invasion (14). The conversion of a static primary tumor into an invasive disseminating metastasis involves an enhanced migratory ability of the tumor cells. Tumor cells use migration mechanisms that are similar, if not identical, to those that occur in normal cells during physiological processes such as embryonic morphogenesis, wound healing, and immune-cell trafficking (10). To migrate, cells must acquire a spatial asymmetry that enables them to turn intracellularly generated forces into net cell body translocation. Dynamic assembly and disassembly of integrin-mediated adhesion and cytoskeletal reorganization are necessary for efficient migration (29). Integrins are heterodimeric integral membrane proteins composed of an α chain and a β chain. Depending on the cell type and extracellular matrix (ECM) substrate, focal contact assembly and migration can be regulated by different integrins. Collagen receptors include α1β1, α2β1, and α3β1 integrins. α1β1 and α3β1 integrins also bind laminin and have less affinity for collagen than does integrin α2β1 (47). The intrinsic propensity of cells to continue migrating in the same direction without turning is closely related to integrin/cytoskeletal interaction, which is known to regulate tractional forces, resulting in modulation of the speed and direction of cell migration (33). Interestingly, different integrin-ECM associations might have opposite effects on the regulation of the directionality of migration. Danen et al. have shown that adhesion to fibronectin by αvβ3 promotes persistent migration through activation of the actin-severing protein cofilin, which results in a polarized phenotype with a single broad lamellipod at the leading edge. In contrast, adhesion to fibronectin by α5β1 instead leads to phosphorylation/inactivation of cofilin and these cells fail to polarize their cytoskeleton and adopt a random/nonpersistent mode of migration (5). Members of the Rho GTPase family (including RhoA, Rac1, and Cdc42) are known as key modulators of cytoskeletal dynamics that occur during cell migration (37). RhoA regulates stress fiber and focal adhesion assembly, Rac regulates the formation of lamellipodial protrusions and membrane ruffles, and Cdc42 triggers filopodial extensions at the cell periphery (13).One of the earliest characterized functions of the Rho GTPase Rac was regulation of the activity of the NADPH oxidase complex in phagocytic cells to produce reactive oxygen species (ROS) (1, 19). Moreover, it has been shown that Rac-dependent ROS production leads to downregulation of RhoA through oxidative inactivation of the low-molecular-weight (LMW) protein tyrosine phosphatase (PTP) and the subsequent activation of p190RhoGAP (31). ROS are also known to directly affect important regulators of cell migration such as PTEN, FAK, or Src (4, 20, 22). ROS are generated in cells from several sources, including the mitochondrial respiratory chain, xanthine oxidase, cytochrome P450, nitric oxide synthase, and NADPH oxidase. The seven known human catalytic subunits of NADPH oxidase include Nox1 to -5 and Duox1 and -2, with Nox2 (gp91phox) being the founding member (21). These oxidases participate in several adaptive functions, ranging from mitogenesis to immune cell signaling (11). A growing body of data points to a key role for ROS production by NADPH oxidase in the control of cell migration and cytoskeletal reorganization (30, 44). Among NADPH oxidase homologs, Nox1 has been detected in different cell types, with major expression in vascular smooth muscle cells and colonic epithelial cells (42). Nox1 involvement in the control of cytoskeletal organization and cellular migration has been only recently reported. Shinohara et al. demonstrated that oncogenic Ras transformation involves Nox1-dependent signaling and leads to inactivation of RhoA. Abrogation of Nox1-dependent ROS production by diphenyleneiodonium (DPI) or small interfering RNA restores RhoA activation and actin stress fiber formation (41). More recently, several groups have highlighted a key role of Nox1 in the control of growth factor-induced migration (16, 38, 40). Cancer cells probably undergo random migration during metastasis, but their migration can be directed by cytokine gradients and/or associated with ECM fibers (29, 55). In a recent report, we showed that Nox1 downregulation decreased the persistence of colonic adenocarcinoma cell migration over collagen I (Col-I) without affecting either the mean velocity or the total distance of migration.In the present study, we investigated the molecular mechanism by which Nox1-dependent ROS production controls the directionality of migration of colonic adenocarcinoma cells. We showed that Nox1-dependent ROS production, which occurs during cell spreading after 4 h of adhesion to Col-I, transiently inhibited RhoA activity. Nox1 inhibition during cell spreading led to a transient increase in cell-matrix contact and initiated a sustained decrease in α2β1 integrin cell surface expression, which was compensated for by an increase in α3 integrin cell surface expression. While Nox1-dependent RhoA inhibition was transient, the observed α2/α3 integrin switch was sustained over 24 h. The loss of directionality observed in cell migration upon Nox1 inhibition may be reversed by α3 integrin blockade. This work shows that Nox1 is involved in the control of integrin surface expression during migration on Col-I, which is critical for persistent directed migration through transient modulation of a RhoA/ROCK-dependent pathway.     

Abl Kinase Inhibits the Engulfment of Apopotic Cells in Caenorhabditis elegans

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor) proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.     

Guiding Neuronal Cell Migrations

Neuronal migration is, along with axon guidance, one of the fundamental mechanisms underlying the wiring of the brain. As other organs, the nervous system has acquired the ability to grow both in size and complexity by using migration as a strategy to position cell types from different origins into specific coordinates, allowing for the generation of brain circuitries. Guidance of migrating neurons shares many features with axon guidance, from the use of substrates to the specific cues regulating chemotaxis. There are, however, important differences in the cell biology of these two processes. The most evident case is nucleokinesis, which is an essential component of migration that needs to be integrated within the guidance of the cell. Perhaps more surprisingly, the cellular mechanisms underlying the response of the leading process of migrating cells to guidance cues might be different to those involved in growth cone steering, at least for some neuronal populations.The migration of newly born neurons is a precisely regulated process that is critical for the development of brain architecture. Neurons arise from the proliferative epithelium that covers the ventricular space throughout the neural tube, an area named the ventricular zone (VZ). From there, newly born neurons adopt two main strategies to disperse throughout the central nervous system (CNS), designated as radial and tangential migration (Hatten 1999; Marín and Rubenstein 2003). During radial migration, neurons follow a trajectory that is perpendicular to the ventricular surface, moving alongside radial glial fibers expanding the thickness of the neural tube. In contrast, tangentially migrating neurons move in trajectories that are parallel to the ventricular surface and orthogonal to the radial glia palisade (Fig. 1). Besides their relative orientation, some of the basic mechanisms underlying the movement of cells using each of these two modes of migration are also different. For example, radially migrating neurons often use radial glial fibers as substrate, whereas tangentially migrating neurons do not seem to require their support to migrate. Even so, neurons may alternate from radial to tangential movement and vice versa during the course of their migration. This suggests that both types of migrations share common principles, in particular those directly related to the cell biology of movement (Marín et al. 2006).Open in a separate windowFigure 1.Representative migrations in the developing CNS. Multiple migrations coexist during embryonic development at different areas of the central nervous system. This schema summarizes some of these migrations during the second week of the embryonic period in the mouse. Neurons use tangential and radial migration to reach their final destination; both strategies are used by the same neurons at different stages of development (i.e., cortical interneurons in the forebrain and precerebellar neurons in the hindbrain). (IML) intermediolateral region of the spinal cord; (IO) inferior olive nucleus; (LGE) lateral ganglionic eminence; (LRN) lateral reticular nucleus; (MGE) medial ganglionic eminence; (NCx) neocortex; (OB) olfactory bulb.One of the structures that better illustrates how both types of migrations are integrated during brain development is the cerebral cortex, and so we will primarily refer to studies performed on cortical neurons for this review. The adult cerebral cortex contains two main classes of neurons: glutamatergic cortical projection neurons (also known as pyramidal cells) and GABAergic interneurons. Pyramidal cells are generated in the ventricular zone (VZ) of the embryonic pallium—the roof of the telencephalon—and reach their final position by radial migration (Rakic 2007). In contrast, cortical interneurons are born in the subpallium—the base of telencephalon—and reach the cerebral cortex through a long tangential migration (Corbin et al. 2001; Marín and Rubenstein 2001).The earliest cortical neurons form a transient structure known as the preplate, around embryonic day 10 (E10) of gestation age in the mouse. This primordial layer consists of Cajal-Retzius cells and the first cohort of pyramidal neurons, which will eventually populate the subplate. Cajal-Retzius cells, which play important roles during neuronal migration, arise from discrete pallial sources and colonize the entire surface of the cortex through tangential migration (Bielle et al. 2005; Takiguchi-Hayashi et al. 2004; Yoshida et al. 2006). The next cohort of pyramidal cells forms the cortical plate (CP) by intercalating in the preplate and splitting this primitive structure in a superficial layer, the marginal zone (MZ or layer I), and a deep layer, the subplate. The development of the neocortex progresses with new waves of neurons that occupy progressively more superficial positions within the CP (Gupta et al. 2002; Marín and Rubenstein 2003). Birth dating studies have shown that layers II–VI of the cerebral cortex are generated in an “inside-out” sequence. Neurons generated earlier reside in deeper layers, whereas later-born neurons migrate past existing layers to form superficial layers (Angevine and Sidman 1961; Rakic 1974). In parallel to this process, GABAergic interneurons migrate to the cortex, where they disperse tangentially via highly stereotyped routes in the MZ, SP, and lower intermediate zone/subventricular zone (IZ/SVZ) (Lavdas et al. 1999). Interneurons then switch from tangential to radial migration to adopt their final laminar position in the cerebral cortex (Ang et al. 2003; Polleux et al. 2002; Tanaka et al. 2003).     

Interfacial instability and the agglutination of erythrocytes by polylysine

Human erythrocytes have been exposed to poylysine of molecular weight range 4 to 220 kDa and concentration range 0.5 to 2,000 /ml at 37°C. Threshold concentrations for cell agglutination by the polycation have been determined for the samples of different molecular weight. Light and electron micrographs show that, in the erythrocyte agglutinates, cell-cell contact is generally made only at discrete, spatially periodic, regions which are distributed over a significant part of the cell surface. The average spacing between contact regions is 0.83 m. The cell membrane has a wavy profile between contact regions. Agglutination occurs only in cell samples whose electrophoretic mobility is significantly altered by polylysine and, in agreement with a previous report, occurs even when the electrophoretic mobility reaches high positive values. The electrophoretic mobility data implies that agglutination requires some protrusion of polylysine from the cell glycocalyx. We discuss how a resulting net attractive intercellular force could act to destabilize the aqueous layer between two cells, allowing surface wave growth which results in spatially periodic contact regions. Examples of situations where cell and membrane contact might be explained by the general concept of interfacial instability are discussed.     

Aligned forces: Origins and mechanisms of cancer dissemination guided by extracellular matrix architecture

Organized extracellular matrix (ECM), in the form of aligned architectures, is a critical mediator of directed cancer cell migration by contact guidance, leading to metastasis in solid tumors. Current models suggest anisotropic force generation through the engagement of key adhesion and cytoskeletal complexes drives contact-guided migration. Likewise, disrupting the balance between cell–cell and cell–ECM forces, driven by ECM engagement for cells at the tumor–stromal interface, initiates and drives local invasion. Furthermore, processes such as traction forces exerted by cancer and stromal cells, spontaneous reorientation of matrix-producing fibroblasts, and direct binding of ECM modifying proteins lead to the emergence of collagen alignment in tumors. Thus, as we obtain a deeper understanding of the origins of ECM alignment and the mechanisms by which it is maintained to direct invasion, we are poised to use the new paradigm of stroma-targeted therapies to disrupt this vital axis of disease progression in solid tumors.     

Extracellular space diffusion and extrasynaptic transmission

The diffusion of neuroactive substances in the extracellular space (ECS) plays an important role in short- and long-distance communication between nerve cells and is the underlying mechanism of extrasynaptic (volume) transmission. The diffusion properties of the ECS are described by three parameters: 1. ECS volume fraction alpha (alpha=ECS volume/total tissue volume), 2. tortuosity lambda (lambda2=free/apparent diffusion coefficient), reflecting the presence of diffusion barriers represented by, e.g., fine neuronal and glial processes or extracellular matrix molecules and 3. nonspecific uptake k'. These diffusion parameters differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Moreover, diffusion barriers may channel the migration of molecules in the ECS, so that diffusion is facilitated in a certain direction, i.e. diffusion in certain brain regions is anisotropic. Changes in the diffusion parameters have been found in many physiological and pathological states in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances in the CNS and thus extrasynaptic transmission, neuron-glia communication, transmitter "spillover" and synaptic cross-talk as well as cell migration, drug delivery and treatment.     

Microbial production of natural δ-decalactone and δ-dodecalactone from the corresponding α,β-unsaturated lactones in Massoi bark oil

Summary The hydrogenation of ,-unsaturated Massoi lactones to natural -lactones by various microorganisms belonging to the Basidiomycetes and by Saccharomyces ce cerevisiae has been studied. Natural -decalactone is an important constituent of several natural flavourings. Process parameters for the hydrogenation by baker's yeast have been characterized on a 2-1 fermentation scale. High hydrogenation activity by baker's yeast was observed at pH 5.5, a temperature of 35° C, no oxygen limitation and constant addition of glucose. By stepwise addition of 2-decen-5-olide about 1.2 g/l of 5-decanolide could be obtained in a fermentation of 16 h. The same concentration could be obtained in 8 h by adding all the substrate at once (1.7 g/l) in the presence of 2% cyclodextrin.Offprint requests to: P. H. van der Schaft     

Migration of the cell nucleus during the amoebo-flagellate transformation ofPhysarum polycephalum is mediated by an actin-generated force that acts on the centrosome

Summary The mechanism of cell-nuclear migration during the amoebo-flagellate transformation inPhysarum polycephalum was examined by fluorescence microscopy after staining with a tubulinspecific antibody, rhodamine-conjugated phalloidin and 4,6-diamidino-2-phenylindole (DAPI). While the round amoeba cells changed to comma-shaped swarm cells within 20min after suspension in buffer, the cell nuclei moved from the central region of each cell to the periphery, each forming a sharp projection in the direction of movement. A centrosome also migrated from the center of the cell to the cell periphery. Since the centrosome was in close contact with the tip that protruded from the cell nucleus throughout the cellnuclear migration, the migration of the cell nucleus and the centrosome could be recognized as comigration. Then the flagella began to elongate from the centrosome and the cells became slender and polarized, adopting the so-called comma-shape. On the basis of these observations, the transformation process was classified into three steps: cell-nuclear migration, flagella formation and swarm maturation. The comigration of the cell nucleus and the centrosome was not inhibited by the anti-microtubule drug nocodazole (4 M) but it was inhibited by the anti-microfilament drug cytochalasin A (4 M), suggesting that the force of migration is generated by microfilaments. To investigate the role of the centrosome in this comigration in detail, we identified two aberrant strains, defective in swimming ability, from among various laboratory strains. The two strains, TM 4 and J, were found to have defects in cell-nuclear migration. Strain TM 4 had two types of irregular swarm cells: in one, only a part of the cell nucleus projected a thin filamentous structure; and in the other, no cell-nuclear migration occurred. Strain J had two centrosomes per cell and such swarm cells exhibited an attempt of cell-nuclear migration at two sites which corresponded to the position of the centrosome. The characteristics of these two strains indicate that the centrosome is essential for cell-nuclear migration. Our observations suggest that the cell-nuclear migration is mediated by actin-generated forces that act on the centrosome rather than on the cell nucleus itself.Abbreviations FITC fluorescein isothiocyanate - DAP 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline - KPB potassium phosphate buffer - MTOC microtubule organizing center     

Intrinsic electric fields and membrane bending

The fragmentation of human erythrocytes heated in a range of ionic environments has been examined by video microscopy, , the average number of surface wave crests growing on the cell rim during fragmentation by membrane externalization, andI, the percentage of cells internalizing membrane, were scored.The membrane diffusion potential was altered experimentally on decreasing the extracellular chloride concentration by substituting either membrane-impermeant sorbitol or Na gluconate for some NaCl. The external-membrane-face surface potential was altered either by surface charge depletion or by ionic strength changes. The dependence of morphological change on diffusion potential at constant cell volume and surface potentials was established over a 34-mV change in diffusion potential. The rate constants for morphological change with charge depletion at different diffusion potentials are largely independent of the diffusion potential. A l.O-mV increase in diffusion potential has an effect on morphological change of comparable magnitude to that of a 1.0-mV decrease in the modulus of the negative surface potential. When the diffusion potential increased on decreasing both the extracellular diffusible ion concentration and extracellular ionic strength, the effect on cell morphology of increasing the modulus of the surface potential was overcome by the effects of the diffusion potential change.     

Relations of the type and branch of surface N-glycans to cell adhesion,migration and integrin expressions

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi -mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [³H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin 51 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated 5, but not 1, and antisense GnT-V decreased both 5 and 1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.     

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×10³–3 × 10³ cells mm^–2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 m diameter and 400 m pitch length (i.e. 10.3% migration at 1 ×10³ cells mm^–2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 m cell numbers were similar to those on the matrix surface: at a depth of 100 m only a few cells were observed.     

Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data

A novel program has been developed for the interpretation of ¹⁵N relaxation rates in terms of macromolecular anisotropic rotational diffusion. The program is based on a highly efficient simulated annealing/minimization algorithm, designed specifically to search the parametric space described by the isotropic, axially symmetric and fully anisotropic rotational diffusion tensor models. The high efficiency of this algorithm allows extensive noise-based Monte Carlo error analysis. Relevant statistical tests are systematically applied to provide confidence limits for the proposed tensorial models. The program is illustrated here using the example of the cytochrome c from Rhodobacter capsulatus, a four-helix bundle heme protein, for which data at three different field strengths were independently analysed and compared.