Base Of Protrusions Research Articles

Misshapen coordinates protrusion restriction and actomyosin contractility during collective cell migration

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.

Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II.

Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell–cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo.

Mutually Inhibitory RAS-PI(3,4)P2 Feedback Loops Mediate Cell Migration

Cell migration mediates a large number of key physiological activities during development and in the adult. These processes require coordination of signal transduction and cytoskeletal events which display excitability, but the mechanisms are poorly understood. Phosphoinositides have played a prominent role in the molecular definition of excitable signal transduction networks. Previously our lab identified a novel Pleckstrin Homology domain containing protein, CynA, which localizes to the lagging edge of cells as well as at the base of protrusions at the front. Here, we find CynA preferentially binds to PI(3,4)P2 on lipid strips and on reconstituted lipid vesicles. Using CynA as a PI(3,4)P2 biosensor, we then show that cells maintain complementary spatial and temporal distributions of Ras activity and PI(3,4)P2 during random migration and in response to chemoattractants. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2 and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive feedback loop essential for excitability. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a novel framework for Ras regulation which may play a key role in many physiological processes.

Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT)

Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton.

Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor.

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.

Traffic jams and shocks of molecular motors inside cellular protrusions.

Molecular motors are involved in key transport processes inside actin-based cellular protrusions. The motors carry cargo proteins to the protrusion tip which participate in regulating the actin polymerization and play a key role in facilitating the growth and formation of such protrusions. It is observed that the motors accumulate at the tips of cellular protrusions and form aggregates that are found to drift towards the protrusion base at the rate of actin treadmilling. We present a one-dimensional driven lattice model, where motors become inactive after delivering their cargo at the tip, or by loosing their cargo to a cargoless neighbor. The results suggest that the experimental observations may be explained by the formation of traffic jams that form at the tip. The model is solved using a novel application of mean-field and shock analysis. We find a new class of shocks that undergo intermittent collapses. Extensions with attachment and detachment events and relevance to experiments are briefly described.

Imaging of a test object with a trapezoidal profile and large side wall inclinations in a scanning electron microscope in the backscattered electron mode

Analysis of imaging of relief pitch structures with trapezoidal profile of relief elements and large side wall inclinations has been carried out in scanning electron microscope in the modes of collection of secondary slow electrons and backscattered electrons. It is shown that the forms of signals of secondary slow electrons and backscattered electrons depend on the structure-element position: close to the pitch-structure edge or far from it. However, the behavior of the signal parameters is independent of the element position. It is demonstrated that the sizes of the parameters of backscattered electron signals characterizing the bottom bases of protrusions and trenches are determined by the true sizes of the bottom bases of the structure elements. At the same time, the sizes of the parameters of the backscattered electron signals that characterize the top bases of protrusions are larger, while those of the trenches are smaller than the sizes determined by the true sizes of the top bases of protrusions and trenches.

Septin 6 regulates the cytoarchitecture of neurons through localization at dendritic branch points and bases of protrusions.

Septins, a conserved family of GTP-binding proteins with a conserved role in cytokinesis, are present in eukaryotes ranging from yeast to mammals. Septins are also highly expressed in neurons, which are post-mitotic cells. Septin6 (SEPT6) forms SEPT2/6/7 complexes in vivo. In this study, we produced a very specific SEPT6 antibody. Immunocytochemisty (ICC) of dissociated hippocampal cultures revealed that SEPT6 was highly expressed in neurons. Developmentally, the expression of SEPT6 was very low until stage 3 (axonal outgrowth). Significant expression of SEPT6 began at stage 4 (outgrowth of dendrites). At this stage, SEPT6 clusters were positioned at the branch points of developing dendrites. In maturing and mature neurons (stage 5), SEPT6 clusters were positioned at the base of filopodia and spines, and pre-synaptic boutons. Detergent extraction experiments also indicated that SEPT6 is not a post-synaptic density (PSD) protein. Throughout morphologic development of neurons, SEPT6 always formed tiny rings (external diameter, ∼0.5 μm), which appear to be clusters at low magnification. When a Sept6 RNAi vector was introduced at the early developmental stage (DIV 2), a significant reduction in dendritic length and branch number was evident. Taken together, our results indicate that SEPT6 begins to be expressed at the stage of dendritic outgrowth and regulates the cytoarchitecture.

Cytoskeletal "jellyfish" structure of Mycoplasma mobile.

Mycoplasma mobile, a parasitic bacterium lacking a peptidoglycan layer, glides on solid surfaces in the direction of a membrane protrusion at a cell pole by a unique mechanism. Recently, we proposed a working model in which cells are propelled by leg proteins clustering at the protrusion's base. The legs repeatedly catch and release sialic acids on the solid surface, a motion that is driven by the force generated by ATP hydrolysis. Here, to clarify the subcellular structure supporting the gliding force and the cell shape, we stripped the membrane by Triton X-100 and identified a unique structure, designated the "jellyfish" structure. In this structure, an oval solid "bell" approximately 235 wide and 155 nm long is filled with a 12-nm hexagonal lattice and connected to this structure are dozens of flexible "tentacles" that are covered with particles of 20-nm diameter at intervals of approximately 30 nm. The particles appear to have 180 degrees rotational symmetry and a dimple at the center. The relation of this structure to the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding. We identified 10 proteins as the components by mass spectrometry and found that these do not show sequence similarities with other proteins of bacterial cytoskeletons or the gliding proteins previously identified. Immunofluorescence and immunoelectron microscopy revealed that two components are localized at the bell and another that has the structure similar to the F(1)-ATPase beta subunit is localized at the tentacles.

Identification of a 123-Kilodalton Protein (Gli123) Involved in Machinery for Gliding Motility of Mycoplasma mobile

Mycoplasma mobile glides on a glass surface in the direction of its tapered end by an unknown mechanism. Two large proteins, Gli349 and Gli521, were recently reported to be involved in glass binding and force generation/transmission, respectively, in M. mobile gliding. These proteins are coded tandemly with two other open reading frames (ORFs) in the order p123-gli349-gli521-p42 on the genome. In the present study, reverse transcriptase PCR analysis suggested that these four ORFs are transcribed cistronically. To characterize the p123 gene coding a 123-kDa protein (Gli123) of 1,128 amino acids, we raised polyclonal antibody against the Gli123 protein. Immunoblotting for Gli123 revealed that Gli123 was missing in a mutant strain, m12, which was previously isolated and characterized by a deficiency in glass binding. Sequencing analysis showed a nonsense mutation at the 523rd amino acid of the protein in the m12 mutant. Immunofluorescence microscopy with the polyclonal antibody showed that Gli123 is localized at the head-like protrusion's base, the cell neck, which is specialized for gliding, as observed for Gli349 and Gli521. Localization of the gliding proteins, Gli349 and Gli521, was disturbed in the m12 mutant, suggesting that Gli123 is essential for the positioning of gliding proteins in the cell neck.

Identification of a 349-kilodalton protein (Gli349) responsible for cytadherence and glass binding during gliding of Mycoplasma mobile.

Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 +/- 200. The antibody inhibited both the hemadsorption and glass binding of M. mobile. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.

FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.

Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.