Changes In Cytoskeletal Organization Research Articles

Neurotrophic extracellular matrix proteins promote neuronal and iPSC astrocyte progenitor cell- and nano-scale process extension for neural repair applications.

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix-based scaffolds for neural repair applications. This study aimed to provide an in-depth analysis of how different commonly used extracellular matrix proteins- laminin-1, Fn, collagen IV, and collagen I-affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)-derived astrocyte progenitors and mouse motor neuron-like cells. Following a 7-day culture period, morphological assessments revealed that laminin-1, fibronectin, and collagen-IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen-IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin-1, fibronectin, and collagen-IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen-I did not enhance neurite growth compared to poly-L-lysine controls. Super-resolution microscopy highlighted extracellular matrix-specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane-associated proteins (laminin-1, fibronectin, and collagen-IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin-1, fibronectin and collagen-IV to promote both iPSC-derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin-1, collagen-IV and fibronectin to elicit strong growth-promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair.

Katanin regulatory subunit B1 (KATNB1) regulates BTB dynamics through changes in cytoskeletal organization.

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured invitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis invivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1)plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology.

Understanding the role of ZO-1 in cell phenotype and gene expression through myosin inhibition

Zonula occludens-1 (ZO-1) is a tight junction protein contributing to junctional formation as well as mediating interactions with junctional proteins and the actomyosin cytoskeleton. To investigate the role of ZO-1, Madin-Darby canine kidney (MDCK) II knockout cells lacking ZO-1 were created utilizing a CRISPR gene editing platform. These ZO-1 KO cells display characteristic phenotypic changes including actin accumulation and junction linearization. We evaluated membrane tension and found ZO-1 loss leads to increased membrane tension and nuclear density. We hypothesized that due changes in cytoskeletal organization and membrane tension the ZO-1 KO cells would spread less following plating. It is suspected that phenotypic changes related to actin accumulation may be the result of myosin dysregulation in the absence of ZO-1. ZO-1 KO cells that are exposed to blebbistatin, a myosin II inhibitor, resemble wild type cells in cell height and junction linearization. RNAseq and qRT-PCR were used to investigate gene expression changes related to ZO-1 loss to further interrogate ZO-1’s role in regulating membrane tension. This study highlights and expands our understanding of how ZO-1 regulates the mechanical properties of epithelial cells. Trinity University, Mach Research Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Plexin-A2 enables the proliferation and the development of tumors from glioblastoma derived cells

The semaphorin guidance factors receptor plexin-A2 transduces sema6A and sema6B signals and may mediate, along with plexin-A4, the anti-angiogenic effects of sema6A. When associated with neuropilins plexin-A2 also transduces the anti-angiogenic signals of sema3B. Here we show that inhibition of plexin-A2 expression in glioblastoma derived cells that express wild type p53 such as U87MG and A172 cells, or in primary human endothelial cells, strongly inhibits cell proliferation. Inhibition of plexin-A2 expression in U87MG cells also results in strong inhibition of their tumor forming ability. Knock-out of the plexin-A2 gene in U87MG cells using CRISPR/Cas9 inhibits cell proliferation which is rescued following plexin-A2 re-expression, or expression of a truncated plexin-A2 lacking its extracellular domain. Inhibition of plexin-A2 expression results in cell cycle arrest at the G2/M stage, and is accompanied by changes in cytoskeletal organization, cell flattening, and enhanced expression of senescence associated β-galactosidase. It is also associated with reduced AKT phosphorylation and enhanced phosphorylation of p38MAPK. We find that the pro-proliferative effects of plexin-A2 are mediated by FARP2 and FYN and by the GTPase activating (GAP) domain located in the intracellular domain of plexin-A2. Point mutations in these locations inhibit the rescue of cell proliferation upon re-expression of the mutated intracellular domain in the knock-out cells. In contrast re-expression of a plexin-A2 cDNA containing a point mutation in the semaphorin binding domain failed to inhibit the rescue. Our results suggest that plexin-A2 may represent a novel target for the development of anti-tumorigenic therapeutics.

Differential modulation of endothelial cytoplasmic protrusions after exposure to graphene-family nanomaterials

Engineered nanomaterials offer the benefit of having systematically tunable physicochemical characteristics (e.g., size, dimensionality, and surface chemistry) that highly dictate the biological activity of a material. Among the most promising engineered nanomaterials to date are graphene-family nanomaterials (GFNs), which are 2-D nanomaterials (2DNMs) with unique electrical and mechanical properties. Beyond engineering new nanomaterial properties, employing safety-by-design through considering the consequences of cell-material interactions is essential for exploring their applicability in the biomedical realm. In this study, we asked the effect of GFNs on the endothelial barrier function and cellular architecture of vascular endothelial cells. Using micropatterned cell pairs as a reductionist in vitro model of the endothelium, the progression of cytoskeletal reorganization as a function of GFN surface chemistry and time was quantitatively monitored. Here, we show that the surface oxidation of GFNs (graphene, reduced graphene oxide, partially reduced graphene oxide, and graphene oxide) differentially affect the endothelial barrier at multiple scales; from the biochemical pathways that influence the development of cellular protrusions to endothelial barrier integrity. More oxidized GFNs induce higher endothelial permeability and the increased formation of cytoplasmic protrusions such as filopodia. We found that these changes in cytoskeletal organization, along with barrier function, can be potentiated by the effect of GFNs on the Rho/Rho-associated kinase (ROCK) pathway. Specifically, GFNs with higher surface oxidation elicit stronger ROCK2 inhibitory behavior as compared to pristine graphene sheets. Overall, findings from these studies offer a new perspective towards systematically controlling the surface-dependent effects of GFNs on cytoskeletal organization via ROCK2 inhibition, providing insight for implementing safety-by-design principles in GFN manufacturing towards their targeted biomedical applications.

HIV-1 Establishes a Sanctuary Site in the Testis by Permeating the BTB Through Changes in Cytoskeletal Organization.

Studies suggest that HIV-1 invades the testis through initial permeation of the blood-testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used 2 approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1-infected Sup-T1 cells to investigate the activity of HIV-1 infection on cocultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin-, and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

Ethanol affects fibroblast behavior differentially at low and high doses: A comprehensive, dose-response evaluation.

This study aims to develop a comprehensive understanding of effects of low and high doses of ethanol on cellular biochemistry and morphology. Here, fibroblast cells are exposed to ethanol of varied concentrations [0.005-10 % (v/v)] to investigate cellular activity, cytoskeletal organization, cellular stiffness, mitochondrial structure, and real-time behavior. Our results indicate a sharp difference in cellular behavior above and below 1 % ethanol concentration. A two-fold increase in MTT activity at low doses is observed, whereas at high doses it decreases. This increased activity at low doses does not involve cell proliferation changes or mitochondrial impairment, as seen at higher doses. Moreover, the study identifies different types of mitochondrial structure impairment at high doses. Morphologically, cells demonstrate a gradual change in cytoskeletal organization and an increase in cell stiffness with increase in doses. Cells exhibit adaptation to sub-toxic doses of ethanol, wherein recovery from ethanol-induced stress is a dose-dependent phenomenon. Cell survival at low doses and toxicity at higher doses are attributed to mild and strong oxidative stress, respectively. Overall, the study provides a comprehensive understanding of dose-dependent effects of ethanol, manifesting its biphasic or hormetic response, biochemically, at low doses and illustrating its toxicological effects at higher doses.

Molecular Defects in Friedreich's Ataxia: Convergence of Oxidative Stress and Cytoskeletal Abnormalities.

Friedreich’s ataxia (FRDA) is a multi-faceted disease characterized by progressive sensory–motor loss, neurodegeneration, brain iron accumulation, and eventual death by hypertrophic cardiomyopathy. FRDA follows loss of frataxin (FXN), a mitochondrial chaperone protein required for incorporation of iron into iron–sulfur cluster and heme precursors. After the discovery of the molecular basis of FRDA in 1996, over two decades of research have been dedicated to understanding the temporal manifestations of disease both at the whole body and molecular level. Early research indicated strong cellular iron dysregulation in both human and yeast models followed by onset of oxidative stress. Since then, the pathophysiology due to dysregulation of intracellular iron chaperoning has become central in FRDA relative to antioxidant defense and run-down in energy metabolism. At the same time, limited consideration has been given to changes in cytoskeletal organization, which was one of the first molecular defects noted. These alterations include both post-translational oxidative glutathionylation of actin monomers and differential DNA processing of a cytoskeletal regulator PIP5K1β. Currently unknown in respect to FRDA but well understood in the context of FXN-deficient cell physiology is the resulting impact on the cytoskeleton; this disassembly of actin filaments has a particularly profound effect on cell–cell junctions characteristic of barrier cells. With respect to a neurodegenerative disorder such as FRDA, this cytoskeletal and tight junction breakdown in the brain microvascular endothelial cells of the blood–brain barrier is likely a component of disease etiology. This review serves to outline a brief history of this research and hones in on pathway dysregulation downstream of iron-related pathology in FRDA related to actin dynamics. The review presented here was not written with the intent of being exhaustive, but to instead urge the reader to consider the essentiality of the cytoskeleton and appreciate the limited knowledge on FRDA-related cytoskeletal dysfunction as a result of oxidative stress. The review examines previous hypotheses of neurodegeneration with brain iron accumulation (NBIA) in FRDA with a specific biochemical focus.

Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro.

Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the “Nichoid”, an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or “2D Nichoid” and multi-layered or “3D Nichoid”) were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells’ specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells’ features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo.

Replicative senescence in MSCWJ-1 human umbilical cord mesenchymal stem cells is marked by characteristic changes in motility, cytoskeletal organization, and RhoA localization

Here, we document changes in cell motility and organization of the contractile apparatus of human umbilical cord Wharton's jelly mesenchymal stem cells (MSCWJ-1) in the process of replicative senescence. Colocalization dynamics of F-actin and actin-binding proteins (myosin-9, α-actinin-4, RhoA) were examined in the MSCWJ-1 cell line. The results show that nuclear-cytoplasmic redistribution of RhoA occurs during replicative senescence, with maximal RhoA/nucleus colocalization evident at passage 15. At that time point, decreases in colocalization, namely myosin-9/F-actin and α-actinin-4/F-actin, were seen and myosin-9 was found in cytosolic extracts in the assembly-incompetent form. Using an automated intravital confocal cytometry system and quantitative analysis of MSCWJ-1 movements, we found that changes in cytoskeletal organization correlate with cell motility characteristics over a time period from passages 9 to 38. The factors examined (cytoskeleton structure, cell motility) indicate that the process by which cells transition to replicative senescence is best represented as three stages. The first stage lasts from cell culture isolation to passage 15 and is characterized by: accumulation of actin-binding proteins in assembly-incompetent forms; nuclear RhoA accumulation; and an increase in movement tortuosity. The second stage extends from passages 15 to 28 and is characterized by: an increase in the structural integrity of the actin cytoskeleton; exit of RhoA and alpha-actinin-4 from the nucleus; and a decrease in path tortuosity. The third stage extends from passage 28 to 38 and is marked by: a plateau in actin cytoskeleton structural integrity; significant decreases in nuclear RhoA levels; and decreases in cell speed.

NC1-peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1.

During the epithelial cycle of spermatogenesis, different sets of cellular events take place across the seminiferous epithelium in the testis. For instance, remodeling of the blood-testis barrier (BTB) that facilitates the transport of preleptotene spermatocytes across the immunological barrier and the release of sperms at spermiation take place at the opposite ends of the epithelium simultaneously at stage VIII of the epithelial cycle. These cellular events are tightly coordinated via locally produced regulatory biomolecules. Studies have shown that collagen α3 (IV) chains, a major constituent component of the basement membrane, release the non-collagenous (NC) 1 domain, a 28-kDa peptide, designated NC1-peptide, from the C-terminal region, via the action of MMP-9 (matrix metalloproteinase 9). NC1-peptide was found to be capable of inducing BTB remodeling and spermatid release across the epithelium. As such, the NC1-peptide is an endogenously produced biologically active peptide which coordinates these cellular events across the epithelium in stage VIII tubules. Herein, we used an animal model, wherein NC1-peptide cloned into the pCI-neo mammalian expression vector was overexpressed in the testis, to better understanding the molecular mechanism by which NC1-peptide regulated spermatogenic function. It was shown that NC1-peptide induced considerable downregulation on a number of cell polarity and planar cell polarity (PCP) proteins, and studies have shown these polarity and PCP proteins modulate spermatid polarity and adhesion via their effects on microtubule (MT) and F-actin cytoskeletal organization across the epithelium. More important, NC1-peptide exerted its effects by downregulating the expression of microtubule (MT) plus-end tracking protein (+TIP) called EB1 (end-binding protein 1). We cloned the full-length EB1 cDNA for its overexpression in the testis, which was found to block the NC1-peptide-mediated disruptive effects on cytoskeletal organization in Sertoli cell epithelium and pertinent Sertoli cell functions. These findings thus illustrate that NC1-peptide is working in concert with EB1 to support spermatogenesis.

Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

In the mammalian testes, such as in rats, the directional alignment of polarized elongating/elongated spermatids, in particular step 17–19 spermatids, across the plane of seminiferous epithelium resembles planar cell polarity (PCP) found in hair cells of the cochlea. It is obvious that spermatid PCP is necessary to support the simultaneous development of maximal number of elongating/elongated spermatids to sustain the daily production of > 50 million sperm per adult rat. Studies have shown that the testis indeed expresses multiple PCP proteins necessary to support spermatid PCP. Herein, using physiological and biochemical assays, and morphological analysis, and with the technique of RNA interference (RNAi) to knockdown PCP protein Dishevelled (Dvl) 1 (Dvl1), Dvl2, Dvl3, or Dvl1/2/3, Dvl proteins, in particular Dvl3, it was shown that Dvl3 played a crucial role of support Sertoli cell tight junction (TJ)-permeability barrier function through changes in the organization of actin- and microtubule (MT)-based cytoskeletons. More important, an in vivo knockdown of Dvl1/2/3 in the testis, defects of spermatid polarity were remarkably noted across the seminiferous epithelium, concomitant with defects of spermatid adhesion and spermatid transport, leading to considerably defects in spermatogenesis. More important, Dvl1/2/3 triple knockdown in the testis also impeded the organization of actin- and MT-based cytoskeletons owing to disruptive spatial expression of actin- and MT-regulatory proteins. In summary, PCP Dishevelled proteins, in particular, Dvl3 is a regulator of Sertoli cell blood–testis barrier (BTB) and also spermatid PCP function through its effects on the actin- and MT-based cytoskeletons in Sertoli cells.

F5-peptide enhances the efficacy of the non-hormonal male contraceptive adjudin

ObjectiveThe bioavailability of the non-hormonal male contraceptive adjudin is low in rats due to the blood-testis barrier (BTB). This study was designed to examine if F5-peptide, an endogenously produced reversible BTB modifier, could enhance the bioavailability of adjudin to affect spermatogenesis and provide a contraceptive effect in rats while reducing systemic toxicity. Study DesignWe overexpressed F5-peptide in adult male rats (n=10 rats; with 3 or 4 rats for each of the three different experiments noted in the three regimens) by intratesticular injection of a mammalian expression vector pCI-neo (pCI-neo/F5-peptide) vs. empty vector alone (pCI-neo/Ctrl) to be followed by treatment with adjudin by oral gavage at a dose of 10 or 20 mg/kg. The status of spermatogenesis was assessed by histological analysis and dual-labeled immunofluorescence analysis on Day 16. To assess fertility, we allowed treated males (n=3–4 rats) to mate with mature female rats (n=3–4) individually, and assessed the number of pups on Days 23, 36 and 82 to assess fertility and reversibility. ResultsAll 4 treated rats overexpressed with F5-peptide and low-dose adjudin were infertile by Day 36, and half of these rats were fertile by Day 82, illustrating reversibility. However, overexpression of F5-peptide alone (or low-dose adjudin alone) had no effects on fertility in n=3 rats. These findings were consistent with the histology data that illustrated the BTB modifier F5-peptide promoted the action of adjudin to induce germ cell exfoliation, mediated by changes in cytoskeletal organization of F-actin and microtubules across the epithelium, thereby reducing the systemic toxicity of adjudin. ConclusionIn this proof-of-concept study, it was shown that overexpression of the F5-peptide prior to administration of adjudin to rats at a low (and ineffective dose by itself) was found to induce reversible male infertility. ImplicationsOverexpression of F5-peptide, an endogenously produced biomolecule in the testis known to induce BTB remodeling, enhanced the contraceptive effect of adjudin in rats, supporting proof of concept studies of BTB disrupters in men.

Role of Periostin in Cardiac Valve Development.

Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.

Bioelectronics of The Cellular Cytoskeleton: Monitoring Cytoskeletal Conductance Variation for Sensing Drug Resistance.

Actin and microtubules form cellular cytoskeletal network, which mediates cell shape, motility and proliferation and are key targets for cancer therapy. Changes in cytoskeletal organization dramatically affect mechanical properties of the cells and correlate with proliferative capacity and invasiveness of cancer cells. Changes in the cytoskeletal network expectedly lead to altered nonmechanical material properties including electrical conductivity as well. Here we applied, for the first time, microtubule and actin based electrical measurement to monitor changes in the electrical properties of breast cancer cells upon administration of anti-tubulin and anti-actin drugs, respectively. Semiconductive behavior of microtubules and conductive behavior of actins presented different bioelectrical responses (in similar frequencies) of the cells treated by anti-tubulin with respect to anti-actin drugs. Doped silicon nanowires were applied as the electrodes due to their enhanced interactive surface and compatibility with electronic fabrication process. We found that treatment with Mebendazole (MBZ), a microtubule destabilizing agent, decreases electrical resistance while treatment with Paclitaxel (PTX), a microtubule stabilizing agent, leads to an increase in electrical resistance. In contrast, actin destabilizing agents, Cytochalasin D (CytD), and actin stabilizing agent, Phalloidin, lead to an increased and decreased electrical resistance, respectively. Our study thus provides proof-of-principle of the usage of determining the electrical function of cytoskeletal compartments in grading of cancer as well as drug resistance assays.

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation.

Characterizing cellular mechanical phenotypes with mechano-node-pore sensing

The mechanical properties of cells change with their differentiation, chronological age, and malignant progression. Consequently, these properties may be useful label-free biomarkers of various functional or clinically relevant cell states. Here, we demonstrate mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell-analysis method that utilizes a four-terminal measurement of the current across a microfluidic channel to quantify simultaneously cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We define a new parameter, the whole-cell deformability index (wCDI), which provides a quantitative mechanical metric of the resistance to compressive deformation that can be used to discriminate among different cell types. The wCDI and the transverse deformation under constant strain show malignant MCF-7 and A549 cell lines are mechanically distinct from non-malignant, MCF-10A and BEAS-2B cell lines, and distinguishes between cells treated or untreated with cytoskeleton-perturbing small molecules. We categorize cell recovery time, ΔTr, as instantaneous (ΔTr~0 ms), transient (ΔTr⩽40 ms), or prolonged (ΔTr>40 ms), and show that the composition of recovery types, which is a consequence of changes in cytoskeletal organization, correlates with cellular transformation. Through the wCDI and cell-recovery time, mechano-NPS discriminates between sub-lineages of normal primary human mammary epithelial cells with accuracy comparable to flow cytometry, but without antibody labeling. Mechano-NPS identifies mechanical phenotypes that distinguishes lineage, chronological age, and stage of malignant progression in human epithelial cells.

TGFβ1-induced expression of caldesmon mediates epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important process that mediates organ development and wound healing, and in pathological contexts, it can contribute to the progression of fibrosis and cancer. During EMT, cells exhibit marked changes in cytoskeletal organization and increased expression of a variety of actin associated proteins. Here, we sought to determine the role of caldesmon in mediating EMT in response to transforming growth factor (TGF)-β1. We find that the expression level and phosphorylation state of caldesmon increase as a function of time following induction of EMT by TGFβ1 and these changes in caldesmon correlate with increased focal adhesion number and size and increased cell contractility. Knockdown and forced expression of caldesmon in epithelial cells reveals that caldesmon expression plays an important role in regulating the expression of the myofibroblast marker alpha smooth muscle actin. Results from these studies provide insight into the role of cytoskeletal associated proteins in the regulation of EMT and may suggest ways to target the cell cytoskeleton for regulating EMT processes.

Varicose and cheerio collaborate with pebble to mediate semaphorin-1a reverse signaling in Drosophila

The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. Here, we uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.

Using Rotary Shadow Electron Microscopy to Characterize Semaphorin-Mediated Growth Cone Collapse.

Rotary shadow electron microscopy (EM) of growth cone cytoskeletons provides a high-resolution method for detecting both global and macromolecular changes in cytoskeletal organization or structure. This approach can be used to study responses to repulsive guidance factors such as semaphorin 3A. Here I describe the procedures used to prepare cultured neurons for rotary-shadow EM, allowing detailed comparisons of cytoskeletal structure.