Cytoskeleton In Response Research Articles

Mechanical stress-induced autophagy is cytoskeleton dependent.

The cytoskeleton is essential for mechanical signal transduction and autophagy. However, few studies have directly demonstrated the contribution of the cytoskeleton to mechanical stress-induced autophagy. We explored the role of the cytoskeleton in response to compressive force-induced autophagy in human cell lines. Inhibition and activation of cytoskeletal polymerization using small chemical molecules revealed that cytoskeletal microfilaments are required for changes in the number of autophagosomes, whereas microtubules play an auxiliary role in mechanical stress-induced autophagy. The intrinsic mechanical properties and special intracellular distribution of microfilaments may account for a large proportion of compression-induced autophagy. Our experimental data support that microfilaments are core components of mechanotransduction signals.

Cooperative interactions between neurotrophin receptors and CXCR4 regulate macrophage phenotype and susceptibility to activation by HIV.

Neural damage due to inflammatory activation of macrophages and microglia is a consequence of HIV infection that leads to cognitive dysfunction. The damage is due, in part, to the release of factors that impair neuronal function but the mechanisms that control their release are poorly understood. Previous studies have shown that mature nerve growth factor (NGF) binding to tropomyosin receptor kinase A (TrkA), and proNGF acting through the p75 neurotrophin receptor (p75NTR) differentially control the phenotype of macrophages in response to HIV. However, the mechanisms responsible for these actions are unclear. The current studies demonstrated that in human monocyte-derived macrophages, CCR5 tropic HIV virions interact with the CXCR4 receptor to promote a neurotoxic macrophage phenotype. TrkA cooperatively interacted with CXCR4 to promote quick and dynamic changes in CXCR4 phosphorylation and more stable downstream actin remodeling in the form of membrane ruffles. TrkA signaling also promoted increased moacrophage calcium spiking, and low neurotoxic activity. Disruption of these interactions by HIV led to an alternative podosome-bearing phenotype with minimal calcium signaling and enhanced toxicity. Neurotrophin receptors provide an independent yet cooperative pathway for modifying the actin cytoskeleton in response to chemokines and subsequent degenerative activity. The strong opposing effects of mature and proneurotrophins may provide the opportunity to develop novel therapies that regulate the phenotype of macrophages in the context of HIV infection and perhaps other degenerative diseases.

Insight to motor clutch model for sensing of ECM residual strain

The mechanical microenvironment strongly affects cell state and decisions. Cell mechanosensing has been described by a molecular clutch which gets progressively engaged depending upon the stiffness of the extracellular material. Through the actuation of pulling forces exerted by actin fibres on the mechanosensitive talin-integrin molecular complex, cells sense and react to the stiffness of their surroundings. However, whether the truly cell mechanosensing is regulated by the pure elastic stiffness or by the strain energy density of the ECM is still debated. Here we report that the cell response to change of strain energy density out of loading induced deformation (purely elastic) can be accounted for by including, within the same frame of the molecular clutch model, the residual strain/stress to which the ECM could be subjected before establishing any interaction with the molecular clutches. To include the contribution of residual stresses, an additional spring orthogonal to the ones already present in the original clutch model has been introduced; this spring takes memory of the ECM strain energy when axially deformed before any interaction with cell molecular clutches can occur. To evaluate the influence of strain on the optimum number of clutches, the model has been implemented with different levels of strain. Results suggest that cells undergo a reinforcement process, stiffening the cytoskeleton in response to the ECM stress/strain energy.

Systems biology approach to understand the interplay between Bacillus anthracis and human host genes that leads to CVDs

Humans infected with invasive Bacillus anthracis (B. anthracis) have a very poor prognosis and are at high risk for developing cardiovascular diseases (CVDs) and shock. Several bacterial elements probably have significant pathogenic roles in this pathogenic process of anthrax. In our current work, we have analysed the molecular level interactions between B. anthracis and human genes to understand the interplay during anthrax that leads to the CVDs. Our results have shown dense interactions between the functional partners in both host and the B. anthracis Gene interaction network (GIN). The functional enrichment analysis indicated that the clusters in the host GIN had genes related to hypoxia and autophagy in response to the lethal toxin; and genes related to adherens junction and actin cytoskeleton in response to edema toxin play a significant role in multiple stages of the disease. The B. anthracis genes BA_0530, guaA, polA, rpoB, ribD, secDF, metS, dinG and human genes ACTB, EGFR, EP300, CTNNB1, ESR1 have shown more than 50 direct interactions with the functional partners and hence they can be considered as hub genes in the network and they are observed to have important roles in CVDs. The outcome of our study will help to understand the molecular pathogenesis of CVDs in anthrax. The hub genes reported in the study can be considered potential drug targets and they can be exploited for new drug discovery.

PS-BPB08-6: REGULATORY MECHANISM OF GLOMERULAR FILTRATION RATE BY THE KEAP1/NRF2 PATHWAY

The activation of the Kelch like ECH associated protein 1 (Keap1)/nuclear factor (erythroid derived 2) like 2 (Nrf2) pathway increases glomerular filtration rate (GFR) in patients with type 2 diabetes and chronic kidney disease. However, the mechanisms whereby the Keap1/Nrf2 pathway regulates GFR are unknown. C57BL/6 mice (WT), Nrf2 deficient mice, and Nrf2 activated Keap1 knockdown mice were compared, and RTA dh404 (CDDO dhTFEA) was used as a Nrf2 activator. Glomerular hemodynamics and the permeability of glomeruli to macromolecules were evaluated using laser Doppler techniques and in vivo imaging. Calcium influx and the rearrangement of the actin cytoskeleton in response to angiotensin II, H2O2, the Nrf2 activator dh404, and transient receptor potential cation channel, subfamily C (TRPC) channel inhibitors were evaluated in podocytes. Finally, the effects of a TRPC6 inhibitor on GFR were investigated in vivo. Pharmacological and genetic Keap1/Nrf2 activation increased renal blood flow (p < 0.05), glomerular volume (p < 0.05), and GFR (p < 0.05), but did not alter afferent or efferent arteriolar diameter or glomerular permeability. Calcium influx into podocytes through TRPC channels in response to angiotensin II or H2O2 was suppressed by Keap1/Nrf2 activation. TRPC inhibitors and dh404 reduced the activation of the small GTPase(RhoA)/Rho associated kinase 1/LIM domain kinase 1/cofilin pathway and the subsequent actin rearrangement induced by angiotensin II or H2O2. Treatment with a TRPC6 inhibitor increased single nephron GFR in WT mice. In conclusion, the Keap1/Nrf2 pathway regulates GFR through changes in ultrafiltration via intracellular calcium signaling and cellular contractility, mediated through TRPC activity, in glomerular cells.

Mechanobiological responses of astrocytes in optic nerve head due to biaxial stretch

BackgroundElevated intraocular pressure (IOP) is the main risk factor for glaucoma, which might cause the activation of astrocytes in optic nerve head. To determine the effect of mechanical stretch on the astrocytes, we investigated the changes in cell phenotype, proteins of interest and signaling pathways under biaxial stretch.MethodThe cultured astrocytes in rat optic nerve head were stretched biaxially by 10 and 17% for 24 h, respectively. Then, we detected the morphology, proliferation and apoptosis of the stretched cells, and performed proteomics analysis. Protein expression was analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. Proteins of interest and signaling pathways were screened using Gene Ontology enrichment analysis and pathway enrichment analysis, and the results were verified by western blot and the gene-chip data from Gene Expression Omnibus (GEO) database.ResultThe results showed that rearrangement of the actin cytoskeleton in response to stimulation by mechanical stress and proliferation rate of astrocytes decreased under 10 and 17% stretch condition, while there was no significant difference on the apoptosis rate of astrocytes in both groups. In the iTRAQ quantitative experiment, there were 141 differential proteins in the 10% stretch group and 140 differential proteins in the 17% stretch group. These proteins include low-density lipoprotein receptor-related protein (LRP6), caspase recruitment domain family, member 10 (CARD10), thrombospondin 1 (THBS1) and tetraspanin (CD81). The western blot results of LRP6, THBS1 and CD81 were consistent with that of iTRAQ experiment. ANTXR2 and CARD10 were both differentially expressed in the mass spectrometry results and GEO database. We also screened out the signaling pathways associated with astrocyte activation, including Wnt/β–catenin pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, ECM-receptor interaction, and transforming growth factor-β (TGF-β) signaling pathway.ConclusionMechanical stimulation can induce changes in cell phenotype, some proteins and signaling pathways, which might be associated with astrocyte activation. These proteins and signaling pathways may help us have a better understanding on the activation of astrocytes and the role astrocyte activation played in glaucomatous optic neuropathy.

An In Vitro Model System to Test Mechano-Microbiological Interactions Between Bacteria and Host Cells.

The aim of this chapter is to present an innovative technique to visualize changes of the F-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust F-actin accumulation in individual cells at the sites where pulling forces were applied. Using the magnetic tweezers, we were able to mimic the local response of the F-actin cytoskeleton to bacteria-generated forces. In this chapter, we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.

Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9.

ABSTRACTIn adaptive immunity, CLEC-2+ dendritic cells (DCs) contact fibroblastic reticular cells (FRCs) inhibiting podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. The molecular mechanisms controlling lymph node remodelling are incompletely understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion, and which other proteins are required for the FRC response to DCs. We find that podoplanin and its partner proteins CD44 and CD9 are differentially expressed by specific lymph node stromal populations in vivo, and their expression in FRCs is coregulated by CLEC-2 (encoded by CLEC1B). Both CD44 and CD9 suppress podoplanin-dependent contractility. We find that beyond contractility, podoplanin is required for FRC polarity and alignment. Independently of podoplanin, CD44 and CD9 affect FRC–FRC interactions. Furthermore, our data show that remodelling of the FRC cytoskeleton in response to DCs is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2 and podoplanin binding inhibits FRC contractility, and, secondly, FRCs form protrusions and spread, which requires both CD44 and CD9. Together, we show a multi-faceted FRC response to DCs, which requires CD44 and CD9 in addition to podoplanin.

Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions.

Cadherin-mediated adhesions (also known as adherens junctions) are adhesive complexes that connect neighboring cells in a tissue. While the role of the actin cytoskeleton in withstanding tension at these sites of contact is well documented, little is known about the involvement of microtubules and the associated endoplasmic reticulum (ER) network in cadherin mechanotransduction. Therefore, we investigated how the organization of ER extensions in close proximity of cadherin-mediated adhesions can affect such complexes, and vice versa. Here, we show that the extension of the ER to cadherin-mediated adhesions is tension dependent and appears to be cadherin-type specific. Furthermore, the different structural organization of the ER/microtubule network seems to affect the localization of ER-bound PTP1B at cadherin-mediated adhesions. This phosphatase is involved in the modulation of vinculin, a molecular clutch which enables differential engagement of the cadherin-catenin layer with the actomyosin cytoskeleton in response to tension. This suggests a link between structural organization of the ER/microtubule network around cadherin-specific adhesions, to control the mechanotransduction of adherens junctions by modulation of vinculin conformational state.

Mechanical compression enhances ciliary beating through cytoskeleton remodeling in human nasal epithelial cells

Nasal inflammatory diseases, including nasal polyps and acute/chronic sinusitis, are characterized by impaired mucociliary clearance and eventually inflammation and infection. Contact of nasal polyps with adjacent nasal mucosa or stagnated mucus within the maxillary sinus produces compressive mechanical stresses on the apical surface of epithelium which can induce cytoskeleton remodeling in epithelial cells. In this study, we hypothesized that compressive stress modulates ciliary beating by altering the mechanical properties of the cytoskeleton of ciliated cell basal bodies. For the primary human nasal epithelial cells, we found that the applied compressive stress higher than the critical value of 1.0 kPa increased the stroke speed of cilia leading to the enhancement of ciliary beating frequency and mucociliary transportability. Immunostained images of the cytoskeleton showed reorganization and compactness of the actin filaments in the presence of compressive stress. Analysis of beating trajectory with the computational modeling for ciliary beating revealed that the stroke speed of cilium increased as the relative elasticity to viscosity of the surrounding cytoskeleton increases. These results suggest that the compressive stress on epithelial cells increases the ciliary beating speed through cytoskeleton remodeling to prevent mucus stagnation at the early stage of airway obstruction. Our study provides an insight into the defensive mechanism of airway epithelium against pathological conditions. Statement of significanceCilia dynamics of the nasal epithelium is critical for not only maintaining normal breathing but preventing inflammatory diseases. It has been shown that mechanical compressive stresses can alter the shape and phenotype of epithelial cells. However, the effect of compressive stress on cilia dynamics is unclear. In this study, we demonstrated that the oscillation speed of cilia in human nasal epithelial cells was increased by the applied compressive stress experimentally. The computational simulation revealed that the change of ciliary beating dynamics was attributed to the viscoelastic properties of the reorganized cytoskeleton in response to compressive stress. Our results will be beneficial in understanding the defensive mechanism of airway epithelium against pathological conditions.

Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

It is well known that living cells interact mechanically with their microenvironment. Many basic cell functions, like migration, proliferation, gene expression, and differentiation, are influenced by external forces exerted on the cell. That is why it is extremely important to study how mechanical properties of the culture substrate influence the cellular molecular regulatory pathways. Optical microscopy is one of the most common experimental method used to visualize and study cellular processes. Confocal microscopy allows to observe changes in the 3D organization of the cytoskeleton in response to a precise mechanical stimulus applied with, for example, a bead trapped with optical tweezers. Optical tweezers-based method (OT) is a microrheological technique which employs a focused laser beam and polystyrene or latex beads to study mechanical properties of biological systems. Latex beads, functionalized with a specific protein, can interact with proteins located on the surface of the cellular membrane. Such interaction can significantly affect the cell’s behavior. In this work, we demonstrate that beads alone, placed on the cell surface, significantly change the architecture of actin, microtubule, and intermediate filaments. We also show that the observed molecular response to such stimulus depends on the duration of the cell–bead interaction. Application of cytoskeletal drugs: cytochalasin D, jasplakinolide, and docetaxel, abrogates remodeling effects of the cytoskeleton. More important, when cells are plated on elastic substrates, which mimic the mechanical properties of physiological cellular environment, we observe formation of novel, “cup-like” structures formed by the microtubule cytoskeleton upon interaction with latex beads. These results provide new insights into the function of the microtubule cytoskeleton. Based on these results, we conclude that rigidity of the substrate significantly affects the cellular processes related to every component of the cytoskeleton, especially their architecture.

EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation.

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.

Mechanisms Linking Mechanotransduction and Cell Metabolism

External forces are sensed by cell surface adhesion receptors, such as E‐cadherin, and trigger robust actin cytoskeletal rearrangements that allow the cell to withstand the force. These actin rearrangements are energetically costly and require approximately half of the ATP in a cell. Previous work in our laboratory demonstrated that in response to force, E‐cadherin signals for enhanced energy production by stimulating AMP‐activated protein kinase (AMPK). AMPK triggers glucose uptake and its oxidation to ATP thereby providing the energy necessary for actin cytoskeletal remodeling. How mechanical force stimulates glucose uptake remains completely unexplored. In this study, we identify glucose transporter‐1 (GLUT1) as a force‐sensitive protein that is highly enriched in the plasma membrane in cells under tension. GLUT1 is necessary for the uptake of glucose and the reinforcement of the actin cytoskeleton that occurs when E‐cadherin experiences force. Intriguingly, GLUT1 is recruited to the cell‐cell junctions and forms a complex with E‐cadherin via a novel linkage mediated by the spectrin adaptor protein, Ankyrin G. Loss of Ankyrin G binding to E‐cadherin inhibits GLUT1 retention to the membrane, glucose uptake, and reinforcement of the actin cytoskeleton in response to force. This study provides the first mechanism for how cell mechanics stimulates the uptake of glucose.Support or Funding InformationThis work is supported by NIH Grant #GM112805.

The Medicago truncatula DREPP Protein Triggers Microtubule Fragmentation in Membrane Nanodomains during Symbiotic Infections.

The initiation of intracellular host cell colonization by symbiotic rhizobia in Medicago truncatula requires repolarization of root hairs, including the rearrangement of cytoskeletal filaments. The molecular players governing microtubule (MT) reorganization during rhizobial infections remain to be discovered. Here, we identified M. truncatula DEVELOPMENTALLY REGULATED PLASMA MEMBRANE POLYPEPTIDE (DREPP), a member of the MT binding DREPP/PCaP protein family, and investigated its functions during rhizobial infections. We show that rhizobial colonization of drepp mutant roots as well as transgenic roots overexpressing DREPP is impaired. DREPP relocalizes into symbiosis-specific membrane nanodomains in a stimulus-dependent manner. This subcellular segregation coincides with DREPP-dependent MT fragmentation and a partial loss of the ability to reorganize the MT cytoskeleton in response to rhizobia, which might rely on an interaction between DREPP and the MT-organizing protein SPIRAL2. Taken together, our results reveal that establishment of symbiotic associations in M. truncatula requires DREPP in order to regulate MT reorganization during initial root hair responses to rhizobia.

Biochemical and immunocytochemical characterization of coronins in platelets

Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets.

P.82First clinical and neuropathological description of a myofibrillar myopathy with congenital onset based on a homozygous recessive FLNC mutation

Filamin C encoded by the FLNC gene is a large actin-cross-linking protein involved in reorganization of actin cytoskeleton in response to signaling events and displaying structural functions at the Z-lines in muscle cells. FLNC-related disorders have been associated with myofibrillar myopathies of adult onset and dominant inheritance. Here, we describe the case of a boy of a consanguineous family with a congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development without cardiac involvement. We performed molecular genetic testing, histopathologic and morphological analyses (including electron microscopy), analysis of the proteomic signature and a FLNC folding assay. Two homozygous mutations (chr7:128478771 C>G; c.1325C>G; homozygous p.Pro442Arg in exon 8) in the FLNC-rod domain were found. The pathogenicity of these mutations could be confirmed by functional analysis. The heterozygous parents are clinically healthy. Based on the mode of inheritance and clinical details, we - for the first time - identified a patient with congenital autosomal recessive myopathy due to a homozygous variant in the FLNC. The disease course started in the neonatal period as a congenital myopathy with development of a distal myopathy in childhood without cardiac involvement, so far. Thus, our combined findings extend the currently recognized clinical and genetic spectrum of filaminopathies.

Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release.

The dynamic response of the cell to osmotic changes is critical to its physiology and is widely exploited for cell manipulation. Here, using three‐dimensional stochastic optical reconstruction microscopy (3D‐STORM), a super‐resolution technique, the hypotonic stress‐induced ultrastructural changes of the cytoskeleton of a common fibroblast cell type are examined. Unexpectedly, these efforts lead to the discovery of a fast, yet reversible dissolution of the vimentin intermediate filament system that precedes ultrastructural changes of the supposedly more dynamic actin and tubulin cytoskeletal systems as well as changes in cell morphology. In combination with calcium imaging and biochemical analysis, it is shown that the vimentin‐specific fast cytoskeletal degradation under hypotonic stress is due to proteolysis by the calcium‐dependent protease calpain. The process is found to be activated by the hypotonic stress‐induced calcium release from intracellular stores, and is therefore efficiently suppressed by inhibiting any part of the IP3‐Ca2+‐calpain pathway established in this study. Together, these findings highlight an unexpected, fast degradation mechanism for the vimentin cytoskeleton in response to external stimuli, and point to the significant, yet previously overlooked physiological impacts of hypotonic stress‐induced intracellular calcium release on cell ultrastructure and function.

Cofilin: Molecular and Cellular Functions and Its Role in the Functioning of the Nervous System

Actin-binding proteins and, in particular, members of the actin-depolymerization factor (ADF)/cofilin family, are involved in the regulation of the actin cytoskeleton in response to various intracellular and extracellular signals. Recent studies point to the exceptional role of this group of proteins in the development and functioning of the nervous system. This review presents the latest data on the functions of cofilin in the cell and the signaling pathways involved in its regulation. Special attention is paid to studies of the relationship between cofilin and actin dynamics in such processes as the control of synaptic plasticity, apoptosis of neurons, and neuroinflammation. We show the molecular mechanisms of cofilin activation-inactivation and the specific structure of actin in nerve cells during neurodegeneration in the in vitro and in vivo models. We review new directions in the study of cofilin and related proteins as prognostic markers and therapeutic targets in the diagnosis and treatment of diseases of the nervous system.

Micro-scaled topographies direct differentiation of human epidermal stem cells

Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. Statement of SignificanceThe epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.

Model for Coordination of Microtubule and Actin Dynamics in Growth Cone Turning

In the developing nervous system, axons are guided to their synaptic targets by motile structures at the axon tip called growth cones, which reorganize their cytoskeleton in order to steer in response to chemotactic cues. Growth cone motility is mediated by an actin-adhesion “clutch” mechanism, in which mechanical attachment to a substrate, coupled with polarized actin growth, produces leading-edge protrusion. Several studies suggest that dynamic microtubules (MTs) in the growth cone periphery play an essential role in growth cone steering. It is not yet well-understood how the MT cytoskeleton and the dynamic actin-adhesion clutch system are coordinated to promote growth cone navigation. I introduce an experimentally motivated stochastic model of the dynamic reorganization of the growth cone cytoskeleton in response to external guidance cues. According to this model, asymmetric decoupling of MTs from actin retrograde flow leads to a local influx of MTs to the growth cone leading edge, and the leading-edge MT accumulation is amplified by positive feedback between MTs and the actin-adhesion clutch system. Local accumulation of MTs at the leading edge is hypothesized to increase actin adhesion to the substrate, which attenuates actin retrograde flow and promotes leading-edge protrusion. Growth cone alignment with the chemotactic gradient is predicted to be most effective for intermediate levels of sensitivity of the adhesion strength to the presence of leading-edge MTs. Quantitative predictions of the MT distribution and the local rate of retrograde actin flow will allow the hypothetical positive feedback mechanism to be experimentally tested.