Adhesion Disassembly Research Articles

Focal adhesions are controlled by microtubules through local contractility regulation

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZ-binding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton. MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing. TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB-231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-β treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions. TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions.

Focal adhesions contain three specialized actin nanoscale layers

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.

S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry.

S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly.

Myosin 1e deficiency affects migration of 4T1 breast cancer cells.

Metastasis of breast cancer cells to distant tissue sites is responsible for the majority of deaths associated with breast cancer. Previously we have examined the role of class I myosin motor protein, myosin 1e (myo1e), in cancer metastasis using the Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) mouse model. Mice deficient in myo1e formed tumors with a more differentiated phenotype relative to the wild-type mice and formed no detectable lung metastases. In the current study, we investigated how the absence of myo1e affects cell migration and invasion in vitro, using the highly invasive and migratory breast cancer cell line, 4T1. 4T1 cells deficient in myo1e exhibited an altered morphology and slower rates of migration in the wound-healing and transwell migration assays compared to the WT 4T1 cells. While integrin trafficking and Golgi reorientation did not appear to be altered upon myo1e loss, we observed lower rates of focal adhesion disassembly in myo1e-deficient cells, which could help explain the cell migration defect.

Force-dependent focal adhesion assembly and disassembly: A computational study.

Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.

HPIP and RUFY3 are noncanonical guanine nucleotide exchange factors of Rab5 to regulate endocytosis-coupled focal adhesion turnover

While the role of endocytosis in focal adhesion turnover-coupled cell migration has been established in addition to its conventional role in cellular functions, the molecular regulators and precise molecular mechanisms that underlie this process remain largely unknown. In this study, we report that proto-oncoprotein hematopoietic PBX-interacting protein (HPIP) localizes to focal adhesions as well as endosomal compartments along with RUN FYVE domain-containing protein 3 (RUFY3) and Rab5, an early endosomal protein. HPIP contains two coiled-coil domains (CC1 and CC2) that are necessary for its association with Rab5 and RUFY3 as CC domain double mutant, that is, mtHPIPΔCC1-2 failed to support it. Furthermore, we show that HPIP and RUFY3 activate Rab5 by serving as noncanonical guanine nucleotide exchange factors of Rab5. In support of this, either deletion of coiled-coil domains or silencing of HPIP or RUFY3 impairs Rab5 activation and Rab5-dependent cell migration. Mechanistic studies further revealed that loss of HPIP or RUFY3 expression severely impairs Rab5-mediated focal adhesion disassembly, FAK activation, fibronectin-associated-β1 integrin trafficking, and thus cell migration. Together, this study underscores the importance of HPIP and RUFY3 as noncanonical guanine nucleotide exchange factors of Rab5 and in integrin trafficking and focal adhesion turnover, which implicates in cell migration.

MAP4K4 regulates forces at cell-cell and cell-matrix adhesions to promote collective cell migration.

Collective cell migration is not only important for development and tissue homeostasis but can also promote cancer metastasis. To migrate collectively, cells need to coordinate cellular extensions and retractions, adhesion sites dynamics, and forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using A431 carcinoma cells, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters, whereas its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell-cell and cell-substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of the actin and plasma membrane crosslinker moesin but disassembles adherens junctions through a moesin-independent mechanism. By analyzing traction and intercellular forces, we found that MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces and the tension loading at the cell-cell adhesions. Together, our results indicate that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration.

Lack of Paxillin phosphorylation promotes single-cell migration in vivo.

Focal adhesions are structures that physically link the cell to the extracellular matrix for cell migration. Although cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish system to visualize focal adhesion structures during single-cell migration in vivo. We find that a key site of phosphoregulation (Y118) on Paxillin exhibits reduced phosphorylation in migrating cells in vivo compared to in vitro. Furthermore, expression of a non-phosphorylatable version of Y118-Paxillin increases focal adhesion disassembly and promotes cell migration in vivo, despite inhibiting cell migration in vitro. Using a mouse model, we further find that the upstream kinase, focal adhesion kinase, is downregulated in cells in vivo, and cells expressing non-phosphorylatable Y118-Paxillin exhibit increased activation of the CRKII-DOCK180/RacGEF pathway. Our findings provide significant new insight into the intrinsic regulation of focal adhesions in cells migrating in their native environment.

Illuminating DEPDC1B in Multi-pronged Regulation of Tumor Progression.

DEPDC1B (aliases BRCC3, XTP8, XTP1) is a DEP (Dishevelled, Egl-1, Pleckstrin) and Rho-GAP-like domains containing predominately membrane-associated protein. Earlier, we and others have reported that DEPDC1B is a downstream effector of Raf-1 and long noncoding RNA lncNB1, and an upstream positive effector of pERK. Consistently, DEPDC1B knockdown is associated with downregulation of ligand-stimulated pERK expression. We demonstrate here that DEPDC1B N-terminus binds to the p85 subunit of PI3K, and DEPDC1B overexpression results in decreased ligand-stimulated tyrosine phosphorylation of p85 and downregulation of pAKT1. Collectively, we propose that DEPDC1B is a novel cross-regulator of AKT1 and ERK, two of the prominent pathways of tumor progression. Our data showing high levels of DEPDC1B mRNA and protein during the G2/M phase have significant implications in cell entry into mitosis. Indeed, DEPDC1B accumulation during the G2/M phase has been associated with disassembly of focal adhesions and cell de-adhesion, referred to as a DEPDC1B-mediated de-adhesion mitotic checkpoint. DEPDC1B is a direct target of transcription factor SOX10, and SOX10-DEPDC1B-SCUBE3 axis has been associated with angiogenesis and metastasis. The Scansite analysis of the DEPDC1B amino acid sequence shows binding motifs for three well-established cancer therapeutic targets CDK1, DNA-PK, and aurora kinase A/B. These interactions and functionalities, if validated, may further implicate DEPDC1B in regulation of DNA damage-repair and cell cycle progression processes. Finally, a survey of the publicly available datasets indicates that high DEPDC1B expression is a viable biomarker in breast, lung, pancreatic and renal cell carcinomas, and melanoma. Currently, the systems and integrative biology of DEPDC1B is far from comprehensive. Future investigations are necessary in order to understand how DEPDC1B might impact AKT, ERK, and other networks, albeit in a context-dependent manner, and influence the actionable molecular, spatial, and temporal vulnerabilities within these networks in cancer cells.

Complexity of progranulin mechanisms of action in mesothelioma

BackgroundMesothelioma is an aggressive disease with limited therapeutic options. The growth factor progranulin plays a critical role in several cancer models, where it regulates tumor initiation and progression. Recent data from our laboratories have demonstrated that progranulin and its receptor, EphA2, constitute an oncogenic pathway in bladder cancer by promoting motility, invasion and in vivo tumor formation. Progranulin and EphA2 are expressed in mesothelioma cells but their mechanisms of action are not well defined. In addition, there are no data establishing whether the progranulin/EphA2 axis is tumorigenic for mesothelioma cells.MethodsThe expression of progranulin in various mesothelioma cell lines derived from all major mesothelioma subtypes was examined by western blots on cell lysates, conditioned media and ELISA assays. The biological roles of progranulin, EphA2, EGFR, RYK and FAK were assessed in vitro by immunoblots, human phospho-RTK antibody arrays, pharmacological (specific inhibitors) and genetic (siRNAs, shRNAs, CRISPR/Cas9) approaches, motility, invasion and adhesion assays. In vivo tumorigenesis was determined by xenograft models. Focal adhesion turnover was evaluated biochemically using focal adhesion assembly/disassembly assays and immunofluorescence analysis with focal adhesion-specific markers.ResultsIn the present study we show that progranulin is upregulated in various mesothelioma cell lines covering all mesothelioma subtypes and is an important regulator of motility, invasion, adhesion and in vivo tumor formation. However, our results indicate that EphA2 is not the major functional receptor for progranulin in mesothelioma cells, where progranulin activates a complex signaling network including EGFR and RYK. We further characterized progranulin mechanisms of action and demonstrated that progranulin, by modulating FAK activity, regulates the kinetic of focal adhesion disassembly, a critical step for cell motility.ConclusionCollectively, our results highlight the complexity of progranulin oncogenic signaling in mesothelioma, where progranulin modulate functional cross-talks between multiple RTKs, thereby suggesting the need for combinatorial therapeutic approaches to improve treatments of this aggressive disease.

Preprint Highlight: Force-dependent recruitment of Piezo1 drives adhesion maturation and calcium entry in normal but not tumor cells.

Piezo1 is a mechanosensitive ion channel implicated in focal adhesion dynamics. This study investigates whether there is direct cross-talk between matrix mechanosensing and Piezo1 channel gating in normal and transformed cells. Live imaging, photobleaching, drug treatments, and gene knockdown approaches in human cell lines are used to characterize Piezo1's role at focal adhesions in 15 cell types. Piezo1 exhibits force-dependent recruitment and activates local calcium signals at focal adhesions to promote adhesion disassembly in normal cells but not in cancer cells. This work establishes a direct connection between two distinct mechanosensing systems, ion channels and cell–matrix adhesions. The role of Piezo1 in processes such as tumor angiogenesis and vascular development underscores the need to mechanistically understand its distinctive functions in normal versus cancer cells.

Fibroblasts secrete fibronectin under lamellipodia in a microtubule- and myosin II-dependent fashion.

Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes β1 integrin-dependent fibrillogenesis. We find that the site of FN secretion is regulated by cell polarization, which occurs in bursts under stabilized lamellipodia at the leading edge. Moreover, analysis of FN secretion and focal adhesion dynamics suggest that focal adhesion formation precedes FN deposition and that deposition continues during focal adhesion disassembly. Lastly, we show that the polarized FN deposition in spreading and migrating cells requires both intact microtubules and myosin II-mediated contractility. Thus, while FN secretion does not require integrin binding, the site of exocytosis is regulated by membrane and cytoskeletal dynamics with secretion occurring after new adhesion formation.

The serine/threonine kinase MINK1 directly regulates the function of promigratory proteins.

Upregulation of the developmental Wnt planar cell polarity (Wnt/PCP) pathway is observed in many cancers and is associated with cancer development. We have recently shown that PRICKLE1, a core Wnt/PCP pathway component, is a marker of poor prognosis in triple-negative breast cancer (TNBC). PRICKLE1 is phosphorylated by the serine/threonine kinase MINK1 and contributes to TNBC cell motility and invasiveness. However, the identity of the substrates of MINK1 and the role of MINK1 enzymatic activity in this process remain to be addressed. We used a phosphoproteomic strategy to identify MINK1 substrates, including LL5β (also known as PHLDB2). LL5β anchors microtubules at the cell cortex through its association with CLASP proteins to trigger focal adhesion disassembly. LL5β is phosphorylated by MINK1, promoting its interaction with CLASP proteins. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in PRICKLE1-LL5β complex assembly and localization, as well as in cell migration. Analysis of gene expression data reveals that the concomitant upregulation of levels of mRNA encoding PRICKLE1 and LL5β, which are MINK1 substrates, is associated with poor metastasis-free survival in TNBC patients. Taken together, our results suggest that MINK1 may represent a potential target for treatment of TNBC.

Merging of ventral fibers at adhesions drives the remodeling of cellular contractile systems in fibroblasts.

Ventral stress fibers (VSFs) are contractile actin fibers dynamically attached to cell-matrix focal adhesions. VSFs are critical in cellular traction force production and migration. VSFs vary from randomly oriented short, thinner fibers to long, thick fibers that span along the whole long axis of a cell. De novo VSF formation was shown to occur by cortical actin mesh condensation or by crosslinking of dorsal stress fibers and transverse arcs at the cell front. However, the formation of long VSFs that extend across the whole cell axis is not well understood. Here, we report a novel phenomenon of VSF merging in migratory fibroblast cells, which is guided by mechanical force balance and contributes to VSF alignment along the long cell axis. The mechanism of VSF merging involves two steps: connection of two ventral fibers by an emerging myosin II bridge at an intervening adhesion and intervening adhesion dissolution. Our data indicate that these two steps are interdependent: slow adhesion disassembly leads to the slowing of the myosin bridge formation. Cellular data and computational modeling show that the contact angle between merging fibers decides successful merging, with shallow angles leading to merge failure. Our data and modeling further show that merging increases the share of uniformly aligned long VSFs, likely contributing to directional traction force production. Thus, we characterize merging as a process for dynamic reorganization of VSFs with functional significance for directional cell migration.

BMP3 inhibits TGFβ2-mediated myofibroblast differentiation during wound healing of the embryonic cornea

Often acute damage to the cornea initiates drastic tissue remodeling, resulting in fibrotic scarring that disrupts light transmission and precedes vision impairment. Very little is known about the factors that can mitigate fibrosis and promote scar-free cornea wound healing. We previously described transient myofibroblast differentiation during non-fibrotic repair in an embryonic cornea injury model. Here, we sought to elucidate the mechanistic regulation of myofibroblast differentiation during embryonic cornea wound healing. We found that alpha-smooth muscle actin (αSMA)-positive myofibroblasts are superficial and their presence inversely correlates with wound closure. Expression of TGFβ2 and nuclear localization of pSMAD2 were elevated during myofibroblast induction. BMP3 and BMP7 were localized in the corneal epithelium and corresponded with pSMAD1/5/8 activation and absence of myofibroblasts in the healing stroma. In vitro analyses with corneal fibroblasts revealed that BMP3 inhibits the persistence of TGFβ2-induced myofibroblasts by promoting disassembly of focal adhesions and αSMA fibers. This was confirmed by the expression of vinculin and pFAK. Together, these data highlight a mechanism to inhibit myofibroblast persistence during cornea wound repair.

Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel.

Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function via tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no in vitro studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical/cellular inhibitory activities. At their tailored EC50 levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC50-levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.

Disassembly of α6β4-mediated hemidesmosomal adhesions promotes tumorigenesis in PTEN-negative prostate cancer by targeting plectin to focal adhesions

Loss of α6β4-dependent hemidesmosomal adhesions has been observed during prostate cancer progression. However, the significance and underlying mechanisms by which aberrant hemidesmosome assembly may modulate tumorigenesis remain elusive. Using an extensive CRISPR/Cas9-mediated genetic engineering approaches in different prostate cancer cell lines combined with in vivo tumorigenesis studies in mice, bone marrow-on-chip assays and bioinformatics, as well as histological analysis of prostate cancer patient cohorts, we demonstrated that simultaneous loss of PTEN and hemidesmosomal adhesions induced several tumorigenic properties including proliferation, migration, resistance to anoikis, apoptosis, and drug treatment in vitro, and increased metastatic capacity in vivo. These effects were plectin-depended and plectin was associated with actin-rich adhesions upon hemidesmosome disruption in PTEN-negative prostate cancer cells leading to activation of EGFR/PI3K/Akt- and FAK/Src-pathways. These results suggest that analysis of PTEN and hemidesmosomal proteins may have diagnostic value helping to stratify prostate cancer patients with high risk for development of aggressive disease and highlight actin-associated plectin as a potential therapeutic target specifically in PTEN/hemidesmosome dual-negative prostate cancer.

Kinetic Changes of Ptdins (3,4,5) P3 within Fast and Slow Turnover Rates of Focal Adhesion.

The assembly and disassembly of the focal adhesions (FA) components occurs throughout life cycle of adhesion, with conservation of balance between removal and recruitment rate during temporal stages. Previous studies have demonstrated that phosphotidyilinositols play a role in regulating FA turnover. However, a little attention has been given to quantify the dynamics changes of Phosphatidylinositol 3,4,5-trisphosphate (PtdIns (3,4,5) P3) within and during fast and slow turnover rates of FA. In this study, we developed a protein purification MDA-MB-231 breast cancer cell line was used as a model in this study due to high metastatic and motile. These cells were co-transfected with GFP- paxillin/vinculin, as FA marker, and the GFP/mCherry-Btk-PH, as a biosensor to visualize PtdIns (3,4,5) P3. Confocal time-lapse images were used to monitor changes or differences in the local generation of PtdIns (3,4,5) P3 within and during assembly and disassembly of FA. Following transfection, immunostaining was used to examine the spatial co-localization between FA and PtdIns (3,4,5) P3. Our data demonstrated that PtdIns (3,4,5) P3 co-localized with FAs and increase during assembly and decline during disassembly of FA which exhibits slow turnover rates and was in a constant level during assembly and disassembly of FA that displays fast turnover rates. Our result suggested that the dynamic changes of PtdIns (3,4,5) P3, it may depend on components undergo turnover, such that early, nascent FA displays fast turnover rates and mature FA exhibits slow turnover rates. Thus, the local enrichment of PtdIns (3,4,5) P3 enhances FA assembly and disassembly activation.