A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase–based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein–dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.     

The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14–MP1 (LAMTOR2/3) complex in FA dynamics. p14–MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end–directed traffic of p14–MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14–MP1 scaffold complex to the vicinity of FAs.     

ARHGAP9 knockdown promotes lung adenocarcinoma metastasis by activating Wnt/β-catenin signaling pathway via suppressing DKK2

This study aims to elucidate the effect of ARHGAP9 on lung adenocarcinoma (LUAD) metastasis, and preliminarily explore its molecular mechanism. As a result, we found that ARHGAP9 was downregulated and correlated with poor prognosis of LUAD. ARHGAP9 knockdown promoted LUAD cell proliferation, migration and invasion, inhibited cell apoptosis and reduced G0G1 cell cycle arrest, in contrast to the results of ARHGAP9 overexpression. Further RNA sequencing analysis demonstrated that ARHGAP9 knockdown in H1299 cells significantly reduced DKK2 (dickkopf related protein 2) expression. Silencing ARHGAP9 in H1299 cells while overexpressing DKK2, DKK2 reversed the promoted effects of ARHGAP9 knockdown on LUAD cell proliferation, migration and invasion. Meanwhile, the activity of Wnt/β-catenin signaling pathway was also reduced. Taken together, these data indicated that ARHGAP9 knockdown promoted LUAD metastasis by activating Wnt/β-catenin signaling pathway via suppressing DKK2. This may provide a new strategy for LUAD treatment.     

Silencing ARHGAP9 correlates with the risk of breast cancer and inhibits the proliferation,migration, and invasion of breast cancer

Breast cancer (BC) is one of the most common malignant tumors in women, and screening relevant genes and markers that are involved in BC tumor genesis and progression is of great value. We previously found that messenger RNA expression of ARHGAP9 was high in BC tissue, but it is unclear whether ARHGAP9 participates in the progression of human BC. In this study, we found that ARHGAP9 expression was correlated with poor patient survival, American Joint Committee on Cancer clinical staging, tumor size, and tumor differentiation. MCF‐7 and MDA‐MB‐231 cells exhibited higher expression of ARHGAP9 than other human BC cell lines (HCC1937, MDA‐MB‐453, ZR‐75‐1, and Hs 578T). Knockdown of ARHGAP9 in human BC cells markedly reduced the cell proliferation, migration, and invasive ability of MCF‐7 and MDA‐MB‐231 cells. Furthermore, small interfering RNA (siRNA) of ARHGAP9 also induced G0‐G1 cell cycle arrest and apoptosis in MCF‐7 and MDA‐MB‐231 cells. Expressions of cell cycle markers (CDK2 and CCNB1) and invasion‐related protein (RhoC and MTA1) were downregulated in siRNA‐ARHGAP9‐transfected cells. siRNA of ARHGAP9 also inhibited the phosphorylation of mitogen‐activated protein kinases in BC cells. In conclusion, the abnormal expression of ARHGAP9 may correlate with the genesis, development, and diagnosis of BC.     

Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

Cell surface receptors integrate chemical and mechanical cues to regulate a wide range of biological processes. Integrin complexes are the mechanotransducers between the extracellular matrix and the actomyosin cytoskeleton. By analogy, cadherin complexes may function as mechanosensors at cell–cell junctions, but this capacity of cadherins has not been directly demonstrated. Furthermore, the molecular composition of the link between E-cadherin and actin, which is needed to sustain such a function, is unresolved. In this study, we describe nanomechanical measurements demonstrating that E-cadherin complexes are functional mechanosensors that transmit force between F-actin and E-cadherin. Imaging experiments reveal that intercellular forces coincide with vinculin accumulation at actin-anchored cadherin adhesions, and nanomechanical measurements show that vinculin potentiates the E-cadherin mechanosensory response. These investigations directly demonstrate the mechanosensory capacity of the E-cadherin complex and identify a novel function for vinculin at cell–cell junctions. These findings have implications for barrier function, morphogenesis, cell migration, and invasion and may extend to all soft tissues in which classical cadherins regulate cell–cell adhesion.     

ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility

Rho GTPases are molecular switches that transmit biochemical signals in response to extracellular stimuli to elicit changes in the actin cytoskeleton. Rho GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. These states are regulated by two distinct families of proteins-guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We studied the role of a previously uncharacterized GAP, ARHGAP18 (MacGAP). Overexpression of ARHGAP18 suppressed the activity of RhoA and disrupted stress fiber formation. Conversely, silencing of ARHGAP18 by small interfering RNA transfection-enhanced stress fiber formation and induced rounding of cells. We examined the role of ARHGAP18 in cell spreading and migration. Immunofluorescence analysis revealed that ARHGAP18 was localized to the leading edge during cell spreading and migration. ARHGAP18-knockdown cells showed impaired spreading, premature formation of stress fibers, and sustained activation of RhoA upon cell attachment. In addition, knockdown and overexpression of ARHGAP18 resulted in the inhibition and promotion of cell migration, respectively. Furthermore, ARHGAP18 was required for the polarization of cells for migration. Our results define ARHGAP18 as one of the crucial factors for the regulation of RhoA for the control of cell shape, spreading, and migration.     

ARHGAP10, a novel human gene coding for a potentially cytoskeletal Rho-GTPase activating protein

Rho-GTPase activating proteins (Rho-GAPs) are negative regulators of Rho-GTPase signaling pathways related to actin cytoskeleton dynamics, cell proliferation, and differentiation. We have identified a novel human gene, termed ARHGAP10, that codes for a 1957-aminoacid Rho-GAP, containing a PDZ, a PH, and a Rho-GAP domain. The cDNA is 7118 bp long and has an open reading frame of 5874 bp. A computational analysis located this gene on chromosome 10 band 10p12.32 suggesting that it is composed of 25 exons. Northern analysis revealed that it is widely expressed, with high levels in brain and muscle. Real-time quantitative PCR analysis confirmed an increase in ARHGAP10 expression during differentiation of HL-60 cells with all-trans-retinoic acid and hematopoietic stem cells with erythropoietin, suggesting that this gene could play a role in normal hematopoiesis. The fact that this gene is highly expressed in muscle and brain, which are highly differentiated tissues, further supports the hypothesis that ARHGAP10 is important for cell differentiation.     

Initiation of focal adhesion assembly by talin and kindlin: A dynamic view

Focal adhesions (FAs) are integrin‐containing protein complexes regulated by a network of hundreds of protein–protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion‐dependent physiological and pathological responses.     

Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration

Rab40b is a SOCS box–containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b–Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b–Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b–Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.     

Modeled microgravity causes changes in the cytoskeleton and focal adhesions, and decreases in migration in malignant human MCF-7 cells

Because cells are sensitive to mechanical forces, microgravity might act on stress-dependent cell changes. Regulation of focal adhesions (FAs) and cytoskeletal activity plays a role in cell maintenance, cell movement, and migration. Human MCF-7 cells were exposed to modeled microgravity (MMG) to test the hypothesis that migration responsiveness to microgravity is associated with cytoskeleton and FA anomalies. MMG acts on MCF-7 cells by disorganizing cytoskeleton filaments (microfilaments and microtubules). Microfilaments in MMG did not display their typical radial array. Likewise, microtubules were disrupted in MCF-7 cells within 4 h of initiation of MMG and were partly reestablished by 48 h. FAs generated in microgravity were less mature than those established in controls, shown by reduced FAs number and clustering. In parallel, MMG decreased kinases activity (such as FAK, PYK2, and ILK) of FAs in MCF-7 cells. The expression of both integrinβ1 and integrinβ4 were downregulated by MMG. We conclude that cytoskeletal alterations and FAs changes in MMG are concomitant with cell invasion and migration retardation. We suggest that reduced migration response in MCF-7 cells following MMG is linked to changes of cytoskeleton and FAs.     

Interleukin-23 Facilitates Thyroid Cancer Cell Migration and Invasion by Inhibiting SOCS4 Expression via MicroRNA-25

Interleukin–23 (IL–23) is a conventional proinflammatory cytokine that plays a role in tumor progression by inducing inflammation in the tumor microenvironment. However, the role of IL–23 in thyroid cancer migration and invasion remains unclear. In the present study, we observed that the treatment with IL–23, induced migration and invasion in human thyroid cancer cells. Additional data demonstrate that SOCS4 negatively regulates IL-23-mediated migration and invasion. On investigating the mechanisms involved in IL–23 mediated migration and invasion, we observed that miR–25 promotes the migration and invasion of thyroid cancer cells by directly binding to the 3′-UTR of SOCS4 that leads to the inhibition of SOCS4. In addition, we also demonstrated that IL–23 increases miR–25 expression levels, and overexpressed miR–25 is involved in IL-23-associated SOCS4 inhibition and cell migration and invasion. Together, our data suggest that IL–23 induces migration and invasion in thyroid cancer cells by mediating the miR–25/SOCS4 signaling pathway.     

The Arf6 GTPase-activating Proteins ARAP2 and ACAP1 Define Distinct Endosomal Compartments That Regulate Integrin α5β1 Traffic

Arf6 and the Arf6 GTPase-activating protein (GAP) ACAP1 are established regulators of integrin traffic important to cell adhesion and migration. However, the function of Arf6 with ACAP1 cannot explain the range of Arf6 effects on integrin-based structures. We propose that Arf6 has different functions determined, in part, by the associated Arf GAP. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin β1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin β1 internalization. Integrin β1 association with adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif (APPL)-positive endosomes and EEA1-positive endosomes was affected by ARAP2 knockdown and depended on ARAP2 GAP activity. ARAP2 formed a complex with APPL1 and colocalized with Arf6 and APPL in a compartment distinct from the Arf6/ACAP1 tubular recycling endosome. In addition, although ACAP1 and ARAP2 each colocalized with Arf6, they did not colocalize with each other and had opposing effects on focal adhesions (FAs). ARAP2 overexpression promoted large FAs, but ACAP1 overexpression reduced FAs. Taken together, the data support a model in which Arf6 has at least two sites of opposing action defined by distinct Arf6 GAPs.     

ZF21 Protein,a Regulator of the Disassembly of Focal Adhesions and Cancer Metastasis,Contains a Novel Noncanonical Pleckstrin Homology Domain

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cellsWe have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, β-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to β-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.     

Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.     

Earlier Migration Timing,Decreasing Phenotypic Variation,and Biocomplexity in Multiple Salmonid Species

Climate-induced phenological shifts can influence population, evolutionary, and ecological dynamics, but our understanding of these phenomena is hampered by a lack of long-term demographic data. We use a multi-decade census of 5 salmonid species representing 14 life histories in a warming Alaskan stream to address the following key questions about climate change and phenology: How consistent are temporal patterns and drivers of phenology for similar species and alternative life histories? Are shifts in phenology associated with changes in phenotypic variation? How do phenological changes influence the availability of resource subsidies? For most salmonid species, life stages, and life histories, freshwater temperature influences migration timing – migration events are occurring earlier in time (mean = 1.7 days earlier per decade over the 3–5 decades), and the number of days over which migration events occur is decreasing (mean = 1.5 days per decade). Temporal trends in migration timing were not correlated with changes in intra-annual phenotypic variation, suggesting that these components of the phenotypic distribution have responded to environmental change independently. Despite commonalities across species and life histories, there was important biocomplexity in the form of disparate shifts in migration timing and variation in the environmental factors influencing migration timing for alternative life history strategies in the same population. Overall, adult populations have been stable during these phenotypic and environmental changes (λ ≈1.0), but the temporal availability of salmon as a resource in freshwater has decreased by nearly 30 days since 1971 due to changes in the median date of migration timing and decreases in intra-annual variation in migration timing. These novel observations advance our understanding of phenological change in response to climate warming, and indicate that climate change has influenced the ecology of salmon populations, which will have important consequences for the numerous species that depend on this resource.     

Beta2-Adaptin Binds Actopaxin and Regulates Cell Spreading,Migration and Matrix Degradation

Cell adhesion to the extracellular matrix is a key event in cell migration and invasion and endocytic trafficking of adhesion receptors and signaling proteins plays a major role in regulating these processes. Beta2-adaptin is a subunit of the AP-2 complex and is involved in clathrin-mediated endocytosis. Herein, β2-adaptin is shown to bind to the focal adhesion protein actopaxin and localize to focal adhesions during cells spreading in an actopaxin dependent manner. Furthermore, β2-adaptin is enriched in adhesions at the leading edge of migrating cells and depletion of β2-adaptin by RNAi increases cell spreading and inhibits directional cell migration via a loss of cellular polarity. Knockdown of β2-adaptin in both U2OS osteosarcoma cells and MCF10A normal breast epithelial cells promotes the formation of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells. These data therefore suggest that actopaxin-dependent recruitment of the AP-2 complex, via an interaction with β2-adaptin, to focal adhesions mediates cell polarity and migration and that β2-adaptin may control the balance between the formation of normal cell adhesions and invasive adhesion structures.