Project description:During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbβ3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.
Project description:Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.
Project description:To generate forces that drive migration of a eukaryotic cell, arrays of actin filaments (F-actin) are assembled at the cell's leading membrane edge. To maintain cell propulsion and respond to dynamic external cues, actin filaments must be disassembled to regenerate the actin monomers (G-actin), and transport of G-actin from sites of disassembly back to the leading edge completes the treadmilling cycle and limits the flux of F-actin assembly. Whether or not molecular diffusion is sufficient for G-actin transport has been a long-standing topic of debate, in part because the dynamic nature of cell motility and migration hinders the estimation of transport parameters. In this work, we applied an experimental system in which cells adopt an approximately constant and symmetrical shape; they cannot migrate but exhibit fast, steady treadmilling in the thin region protruding from the cell. Using fluorescence recovery after photobleaching, we quantified the relative concentrations and corresponding fluxes of F- and G-actin in this system. In conjunction with mathematical modeling, constrained by measured features of each region of interest, this approach revealed that diffusion alone cannot account for the transport of G-actin to the leading edge. Although G-actin diffusion and vectorial transport might vary with position in the protruding region, good agreement with the fluorescence recovery after photobleaching measurements was achieved by a model with constant G-actin diffusivity ∼2 μm2/s and anterograde G-actin velocity less than 1 μm/s.
Project description:During cell motion on a substratum, eukaryotic cells project sheetlike lamellipodia which contain a dynamically remodeling three-dimensional actin mesh. A number of regulatory proteins and subtle mechano-chemical couplings determine the lamellipodial protrusion dynamics. To study these processes, we constructed a microscopic physico-chemical computational model, which incorporates a number of fundamental reaction and diffusion processes, treated in a fully stochastic manner. Our work sheds light on the way lamellipodial protrusion dynamics is affected by the concentrations of actin and actin-binding proteins. In particular, we found that protrusion speed saturates at very high actin concentrations, where filament nucleation does not keep up with protrusion. This results in sparse filamentous networks, and, consequently, high resistance forces on individual filaments. We also observed maxima in lamellipodial growth rates as a function of Arp2/3, a nucleating protein, and capping proteins. We provide detailed physical explanations behind these effects. In particular, our work supports the actin-funneling-hypothesis explanation of protrusion speed enhancement at low capping protein concentrations. Our computational results are in agreement with a number of related experiments. Overall, our work emphasizes that elongation and nucleation processes work highly cooperatively in determining the optimal protrusion speed for the actin mesh in lamellipodia.
Project description:Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.
Project description:Actin-based cellular protrusions are a ubiquitous feature of cell morphology, e.g., filopodia and microvilli, serving a huge variety of functions. Despite this, there is still no comprehensive model for the mechanisms that determine the geometry of these protrusions. We present here a detailed computational model that addresses a combination of multiple biochemical and physical processes involved in the dynamic regulation of the shape of these protrusions. We specifically explore the role of actin polymerization in determining both the height and width of the protrusions. Furthermore, we show that our generalized model can explain multiple morphological features of these systems, and account for the effects of specific proteins and mutations.
Project description:Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatidylinositol 3,4,5-triphosphate (PIP(3)), two phosphoinositides that play important roles in cell signaling. Binding is not Ca(2+)-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP(2) and PIP(3) in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP(2) localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.
Project description:Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin alpha(2)beta(1), glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.
Project description:The four types of platelet-derived growth factors (PDGFs) and the two types of PDGF receptors (PDGFRs, which belong to class III receptor tyrosine kinases) have important functions in the development of connective tissue cells. Recent structural studies have revealed novel mechanisms of PDGFs in propeptide loading and receptor recognition/activation. The detailed structural understanding of PDGF-PDGFR signaling has provided a template that can aid therapeutic intervention to counteract the aberrant signaling of this normally silent pathway, especially in proliferative diseases such as cancer. This review summarizes the advances in the PDGF system with a focus on relating the structural and functional understandings, and discusses the basic aspects of PDGFs and PDGFRs, the mechanisms of activation, and the insights into the therapeutic antagonism of PDGFRs. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.
Project description:Cadherin-mediated cell-cell adhesion is actin-dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell-cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.