Induced Formation of Plasma Membrane Protrusions with Porous Materials as Instructive Surfaces.

The plasma membrane is a vital component in cellular processes, and its structure has a significant impact on cellular behavior. The physical characteristics of the extracellular environment, along with the presence of surface pores, can influence the formation of membrane protrusions. Nanoporous surfaces have demonstrated their capacity to induce membrane protrusions in both adherent and non-adherent cells. This chapter presents a methodology that utilizes a nanoporous substrate with nanotopographical constraints to effectively stimulate the formation of membrane protrusions in cells.

Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses.

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.

Impairment of Assembly of the Vimentin Intermediate Filaments Leads to Suppression of Formation and Maturation of Focal Contacts and Alteration of the Type of Cellular Protrusions.

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by formation of lamellipodia and/or filopodia, while amoeboid migration is based on bleb formation. Changing of migrational conditions can lead to alteration in the character of cell movement. For example, inhibition of the Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility mode. Ability of the cells to switch from one type of motility to another is called migratory plasticity. Cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryo fibroblasts REF52 with normal VIF organization, fibroblasts with vimentin knockout (REF-/-), and fibroblasts with mutation inhibiting assembly of the full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase in the number of cells with blebs. The fibroblasts with short fragments of vimentin demonstrate the significant increase in number of cells forming blebs both spontaneously and in the presence of CK-666. Disruption of the VIF organization did not lead to the significant changes in the microtubules network or the level of myosin light chain phosphorylation, but caused significant reduction in the focal contact system. The most pronounced and statistically significant decrease in both size and number of focal adhesions were observed in the REF117 cells. We believe that regulation of the membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that organization of VIFs determines the type of cell protrusions, which, in turn, determines the character of cell movement. A novel role of VIFs as a regulator of membrane blebbing, essential for manifestation of the migratory plasticity, is shown.

SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.

LncRNAs are emerging as important regulators of gene expression by controlling transcription in the nucleus and by modulating mRNA translation in the cytoplasm. In this study, we reveal a novel function of lncRNA SNHG15 in mediating breast cancer cell invasion through regulating the local translation of CDH2 mRNA. We show that SNHG15 preferentially localizes at the cellular protrusions or cell leading edge and that this localization is directed by IMP1, a multifunctional protein involved in many aspects of RNA regulation. We demonstrate that SNHG15 also forms a complex with nucleolin, allowing nucleolin to be co-transported with SNHG15 to the cell protrusions, where the accumulated nucleolin is able to bind to CDH2 mRNA. Interaction with nucleolin stabilizes local CDH2 mRNA and regulates its translation, thus promoting cell invasive potential. Our findings reveal an underlying mechanism by which lncRNA could serve as a carrier to transport a protein regulator into a specific cell compartment to enhance target mRNA expression.

Reciprocal intra- and extra-cellular polarity enables deep mechanosensing through layered matrices.

Adherent cells migrate on layered tissue interfaces to drive morphogenesis, wound healing, and tumor invasion. Although stiffer surfaces are known to enhance cell migration, it remains unclear whether cells sense basal stiff environments buried under softer, fibrous matrix. Using layered collagen-polyacrylamide gel systems, we unveil a migration phenotype driven by cell-matrix polarity. Here, cancer (but not normal) cells with stiff base matrix generate stable protrusions, faster migration, and greater collagen deformation because of "depth mechanosensing" through the top collagen layer. Cancer cell protrusions with front-rear polarity produce polarized collagen stiffening and deformations. Disruption of either extracellular or intracellular polarity via collagen crosslinking, laser ablation, or Arp2/3 inhibition independently abrogates depth-mechanosensitive migration of cancer cells. Our experimental findings, validated by lattice-based energy minimization modeling, present a cell migration mechanism whereby polarized cellular protrusions and contractility are reciprocated by mechanical extracellular polarity, culminating in a cell-type-dependent ability to mechanosense through matrix layers.

Blebs promote cell survival by assembling oncogenic signalling hubs.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

Two Rac1 pools integrate the direction and coordination of collective cell migration.

Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

Cellular protrusions in 3D: Orchestrating early mouse embryogenesis.

Cellular protrusions generated by the actin cytoskeleton are central to the process of building the body of the embryo. Problems with cellular protrusions underlie human diseases and syndromes, including implantation defects and pregnancy loss, congenital birth defects, and cancer. Cells use protrusive activity together with actin-myosin contractility to create an ordered body shape of the embryo. Here, I review how actin-rich protrusions are used by two major morphological cell types, epithelial and mesenchymal cells, during collective cell migration to sculpt the mouse embryo body. Pre-gastrulation epithelial collective migration of the anterior visceral endoderm is essential for establishing the anterior-posterior body axis. Gastrulation mesenchymal collective migration of the mesoderm wings is crucial for body elongation, and somite and heart formation. Analysis of mouse mutants with disrupted cellular protrusions revealed the key role of protrusions in embryonic morphogenesis and embryo survival. Recent technical approaches have allowed examination of the mechanisms that control cell and tissue movements in vivo in the complex 3D microenvironment of living mouse embryos. Advancing our understanding of protrusion-driven morphogenesis should provide novel insights into human developmental disorders and cancer metastasis.

Removal of cellular protrusions.

Cell-cell communications are central to a variety of physiological and pathological processes in multicellular organisms. Cells often rely on cellular protrusions to communicate with one another, which enable highly selective and efficient signaling within complex tissues. Owing to significant improvements in imaging techniques, identification of signaling protrusions has increased in recent years. These protrusions are structurally specialized for signaling and facilitate interactions between cells. Therefore, physical regulation of these structures must be key for the appropriate strength and pattern of signaling outcomes. However, the typical approaches for understanding signaling regulation tend to focus solely on changes in signaling molecules, such as gene expression, protein-protein interaction, and degradation. In this short review, we summarize the studies proposing the removal of different types of signaling protrusions-including cilia, neurites, MT (microtubule based)-nanotubes and microvilli-and discuss their mechanisms and significance in signaling regulation.

The type 3 secretion effector IpgD promotes S. flexneri dissemination.

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.

The core autophagy protein ATG9A controls dynamics of cell protrusions and directed migration.

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)-rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of ß1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix.

ALFY localizes to early endosomes and cellular protrusions to facilitate directional cell migration.

Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility.

Quantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy.

Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.

Chronic arsenic increases cell migration in BEAS-2B cells by increasing cell speed, cell persistence, and cell protrusion length.

There is a strong association between arsenic exposure and lung cancer development, however, the mechanism by which arsenic exposure leads to carcinogenesis is not clear. In our previous study, we observed that when BEAS-2B cells are chronically exposed to arsenic, there is an increase in secreted TGFα, as well as an increase in EGFR expression and activity. Further, these changes were broadly accompanied with an increase in cell migration. The overarching goal of this study was to acquire finer resolution of the arsenic-dependent changes in cell migration, as well as to understand the role of increased EGFR expression and activity levels in the underlying mechanisms of cell migration. To do this, we used a combination of biochemical and single cell assays, and observed chronic arsenic treatment enhancing cell migration by increasing cell speed, cell persistence and cell protrusion length. All three parameters were further increased by the addition of TGFα, indicating EGFR activity is sufficient to enhance those aspects of cell migration. In contrast, EGFR activity was necessary for the increase in cell speed, as it was reversed with an EGFR inhibitor, AG1478, but was not necessary to enhance persistence and protrusion length. From these data, we were able to isolate both EGFR-dependent and -independent features of cell migration that were enhanced by chronic arsenic exposure.

Shigella flexneri subverts host polarized exocytosis to enhance cell-to-cell spread.

Shigella flexneri is a gram-negative bacterial pathogen that causes dysentery. Critical for disease is the ability of Shigella to use an actin-based motility (ABM) process to spread between cells of the colonic epithelium. ABM transports bacteria to the periphery of host cells, allowing the formation of plasma membrane protrusions that mediate spread to adjacent cells. Here we demonstrate that efficient protrusion formation and cell-to-cell spread of Shigella involves bacterial stimulation of host polarized exocytosis. Using an exocytic probe, we found that exocytosis is locally upregulated in bacterial protrusions in a manner that depends on the Shigella type III secretion system. Experiments involving RNA interference (RNAi) indicate that efficient bacterial protrusion formation and spread require the exocyst, a mammalian multi-protein complex known to mediate polarized exocytosis. In addition, the exocyst component Exo70 and the exocyst regulator RalA were recruited to Shigella protrusions, suggesting that bacteria manipulate exocyst function. Importantly, RNAi-mediated depletion of exocyst proteins or RalA reduced the frequency of protrusion formation and also the lengths of protrusions, demonstrating that the exocyst controls both the initiation and elongation of protrusions. Collectively, our results reveal that Shigella co-opts the exocyst complex to disseminate efficiently in host cell monolayers.

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions.

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport.

Pyk2 regulates cell-edge protrusion dynamics by interacting with Crk.

Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2-/- mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2-/- MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein-protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2-/- MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand.

A novel method for successful induction of interdigitating process formation in conditionally immortalized podocytes from mice, rats, and humans.

Formation of processes in podocytes is regarded as the hallmark of maturity and normal physical condition for the cell. There are many accumulated findings about molecular mechanisms that cause retraction of podocyte processes; however, there is little knowledge of the positive mechanisms that promote process formation in vitro, and most previous reports about this topic have been limited to low-density cultures. Here, we found that process formation can be induced in 100% confluent cultures of conditionally immortalized podocytes in mouse, rat, and human species by combining serum depletion and Y-27632 ROCK inhibitor supplementation on the scaffold of laminin-521(L521). We noted the cytoskeletal reorganization of the radial extension pattern of vimentin filaments and downregulation of actin stress fiber formation under that condition. We also found that additional standard amount of serum, depletion of ROCK inhibitor, or slight mismatch of the scaffold as laminin-511(L511) hinder process formation. These findings suggest that the combination of reduced serum, podocyte-specific scaffold, and intracellular signaling to reduce the overexpression of ROCK are required factors for process formation.

The GAS6-AXL signaling pathway triggers actin remodeling that drives membrane ruffling, macropinocytosis, and cancer-cell invasion.

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.