Jonathan Winkelman - Academia.edu (original) (raw)

Papers by Jonathan Winkelman

Cell-cell fusion is an indispensable process in the conception, development and physiology of mul... more Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamin's proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically stiff actin network. Thus, dynamin functions as a unique actin-bundling protein that enhances mechanical force generation by the F-actin network in a GTPase-dependent manner. Our findings have universal implications for understanding dynamin-actin interactions in various cellular processes beyond cell-cell fusion.

Cytoskeleton, Jun 1, 2021

This is the author manuscript accepted for publication and has undergone full peer review but has... more This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as

Proceedings of the National Academy of Sciences of the United States of America, Sep 28, 2020

The actin cytoskeleton assembles into diverse load-bearing networks including stress fibers, musc... more The actin cytoskeleton assembles into diverse load-bearing networks including stress fibers, muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl-1 & Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force-sensitivity. Zyxin rapidly localizes via its LIM domains to failing stress fibers in cells, known as strain sites, to initiate stress fiber repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We observe that many, but not all, LIM domains from functionally diverse proteins localize to spontaneous or induced stress fiber strain sites in mammalian cells. Additionally, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to stress fiber strain sites in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. Sequence analysis and mutagenesis of the LIM domain region of zyxin indicate a requirement of tandem LIM domains, which contribute additively, for sensing stress fiber strain sites. In vitro, purified LIM domains from mammalian zyxin and fission yeast Pxl1 bind to mechanically stressed F-actin networks but do not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress. .

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2015

Cells contain multiple F-actin assembly pathways including the Arp2/3 complex, formins, and Ena/V... more Cells contain multiple F-actin assembly pathways including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of Factin from a common pool of G-actin; however, the interplay/competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2−/− cells lack lamellipodia and migrate slower than WT cells, but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2−/− cells was resistant to cytochalasin-D and was highly

Proceedings of the National Academy of Sciences of the United States of America, Mar 3, 2014

Nature Cell Biology, May 25, 2020

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and... more The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12–16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Journal of Biological Chemistry, Jul 1, 2011

In cells, actin binding proteins (ABPs) sort to different regions in order to establish F-actin n... more In cells, actin binding proteins (ABPs) sort to different regions in order to establish F-actin networks with diverse functions, including filopodia used for cell migration, or contractile rings required for cell division. Recent experimental work uncovered a passive mechanism that may facilitate spatial localization of ABPs: binding of a short crosslinker protein to two actin filaments promotes the binding of other short crosslinkers and inhibits the binding of longer crosslinkers (and vice versa). We hypothesize this sorting arises because F-actin is semiflexible and cannot bend over short distances. We develop a mathematical theory and a kinetic Monte Carlo simulation encompassing the most important physical parameters for this process, and use simulations of a coarse-grained but molecularly explicit model to characterize and test our predictions about the interplay of mechanical and kinetic parameters. Our theory and data predict an explicit dependence of crosslinker separation on bundle polymerization rate. We perform experiments that confirm a dependence on polymerization rate, but in an unanticipated non-monotonic manner. We use simulations to show that this non-monotonic behavior can arise in situations where crosslinkers have equal bundling affinity at equilibrium, but differing microscopic binding rates to filaments. This dependence of sorting on actin polymerization rate is a non-equilibrium effect, qualitatively similar to non-equilibrium domain formation in materials growth. Thus our results reveal an avenue by which cells can organize molecular material to drive biological processes, and can also guide the choice and design of crosslinkers for engineered protein-based materials. Cytoskeleton | Sorting | Modeling Networks formed from filamentous actin polymers (F-actin) perform diverse mechanical tasks throughout cells, such as enabling migration (1, 2), adhesion (3), mechanosensing (4) and division (5). F-actin is formed into a network by crosslinkers, actin binding proteins (ABPs) that link multiple filaments. A large variety of crosslinkers exist, with diverse kinetic and mechanical properties (5). For example, the actin crosslinker fimbrin can bundle branched F-actin at the leading edge of migrating cells so that they can harness energy from actin polymerization to generate protrusive forces (1, 6). The force propagating F-actin cables that maintain a cell's shape, or which are contained within a cytokinetic ring, each use their own F-actin crosslinking protein to form a specific geometry (7). Many ABPs may be involved in one single cellular mechanism. The cytokinetic ring of fission yeast employs formins to assemble F-actin, the crosslinker α-actinin to connect F-actin into anti-parallel bundles, and myosins to contract the bundles and ultimately divide the cell (8, 9). ABP kinetics can play subtle roles in these processes. For example, we previously showed that having optimal kinetics of binding (kon, k off), in addition to a an optimal binding affinity (K d = k off /kon) for the crosslinker α-actinin is crucial for proper contractile ring formation and constriction during cell division (10). Regulating the spatial and temporal organization of ABPs in a crowded cellular environment is understandably complex, and determining the mechanisms involved is an active area of research. Some of this regulation may require explicit signaling pathways; for example generation of branched networks by the Arp2/3 complex can be activated by upstream activation of a Rho GTPase (11, 12). In addition to these signaling-based mechanisms, emerging data detail many passive mechanisms by which competition between different components for the same substrate can allow self-regulation and localization of ABPs in the actin cytoskeleton (13-16). We recently showed that α-actinin and fascin, two F-actin crosslinkers that are primarily found separated into different F-actin networks within cells, can self-sort in a simplified in vitro reconstitution of a branched Arp2/3 complex-nucleated network, and even sort to different domains within the same two-filament actin bundle (Figure 1A) (15). An outstanding challenge is to determine which of the biochemical characteristics of actin, fascin, and α-actinin yield sorting, and in that way determine if this mechanism may be generalizable to other polymers or crosslinkers. An important difference between fascin and α-actinin is their size; fascin is small (∼8 nm), and therefore forms tight bundles composed of narrowly-spaced actin filaments, while α-actinin is larger (∼35 nm) and therefore forms bundles composed of actin filaments that are more widely spaced (15, 17, 18). While filaments in α-actinin bundles are arranged with mixed polarity, fascin assembles bundles composed exclusively of parallel filaments, such that their fast-growing barbed ends all face the same direction (19, 20). Therefore the structures observed in our previous work (such as the one shown in Figure 1A) are parallel two-filament bundles in which the spacing between filaments alternates between approximately 8 and 35 nm (15). For transitions in bundle spacing, the actin filaments must bend significantly over length scales shorter than their persistence length Lp = 17 µm (21), which is energetically unfavorable. Since we observe domains in experiment, the energetic cost of bending must be compensated by favorable effects, such as the benefit of binding more crosslinkers and the entropic gain of mixing components on the bundle. In this work, we use this hypothesis to develop a theoretical model that enables investigating the full range of mechanical

Current Biology, Dec 1, 2022

Cytoskeleton, 2021

The actin cytoskeleton is important for maintaining mechanical homeostasis in adherent cells, lar... more The actin cytoskeleton is important for maintaining mechanical homeostasis in adherent cells, largely through its regulation of adhesion and cortical tension. The LIM (Lin‐11, Isl1, MEC‐3) domain‐containing proteins are involved in a myriad of cellular mechanosensitive pathways. Recent work has discovered that LIM domains bind to mechanically stressed actin filaments, suggesting a novel and widely conserved mechanism of mechanosensing. This review summarizes the current state of knowledge of LIM protein mechanosensitivity.

During embryonic morphogenesis, the integrity of epithelial tissues depends on the ability of cel... more During embryonic morphogenesis, the integrity of epithelial tissues depends on the ability of cells in tissue sheets to undergo rapid changes in cell shape while preventing self-injury to junctional actin networks. LIM domain-containing repeat (LCR) proteins are recruited to sites of strained actin filaments in cultured cells, and are therefore promising candidates for mediating self-healing of actin networks, but whether they play similar roles in living organisms has not been determined. Here, we establish roles for Caenorhabditis elegans TES-1/Tes, an actin-binding LCR protein present at apical junctions, during epithelial morphogenesis. TES-1∷GFP is recruited to apical junctions during embryonic elongation, when junctions are under tension; in embryos in which stochastic failure of cell elongation occurs, TES-1 is only strongly recruited to junctions in cells that successfully elongate, and recruitment is severely compromised in genetic backgrounds in which cell shape changes do...

Nature Cell Biology, 2020

Cell-cell fusion is an indispensable process in the conception, development and physiology of mul... more Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamin’s proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically...

Current Biology, 2016

Highlights d Purified bundling proteins intrinsically sort within reconstituted actin networks d ... more Highlights d Purified bundling proteins intrinsically sort within reconstituted actin networks d Fascin and a-actinin segregate to F-actin bundles with different filament spacing d Fascin and a-actinin facilitate their own binding while inhibiting the other d The compact bundlers fascin, espin, and fimbrin cosegregate away from a-actinin

Science (New York, N.Y.), Jan 31, 2015

The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, in... more The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin cross-linking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here, we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently "poisoned" the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by using actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses.

Developmental Cell, 2015

Cells contain multiple F-actin assembly pathways, including the Arp2/3 complex, formins, and Ena/... more Cells contain multiple F-actin assembly pathways, including the Arp2/3 complex, formins, and Ena/ VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay and/or competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2 À/À cells lack lamellipodia and migrate more slowly than WT cells but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2 À/À cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. Antagonism of the Arp2/3 complex by profilin-1 in cells appears to maintain actin homeostasis by balancing Arp2/3 complex-dependent and-independent actin assembly pathways.

Proceedings of the National Academy of Sciences, 2014

Significance Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are required for t... more Significance Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are required for the formation and maintenance of filopodia, finger-like projections at the leading edge of migrating cells that are composed of parallel actin filaments bundled by Fascin. We imaged individual fluorescently labeled Drosophila Ena molecules on both single and Fascin-bundled actin filaments in vitro. Ena stimulates actin assembly by remaining continuously associated with the barbed end and increasing the elongation rate by approximately two- to threefold. Remarkably, the frequency and length of Ena’s processive runs are enhanced on filaments within a Fascin bundle, which drives a positive feedback cycle that allows the assembly of uniformly thick filopodia-like F-actin bundles composed of multiple filaments with aligned ends.

Nature Structural & Molecular Biology, 2011

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand the... more Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio VopL depends on its three W domains and dimerization through a unique VopL Cterminal domain (VCD). The VCD displays a novel all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment, but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

Microbial Ecology, 2012

The concentration of CO 2 in the Earth's atmosphere has increased over the last century. Although... more The concentration of CO 2 in the Earth's atmosphere has increased over the last century. Although this increase is unlikely to have direct effects on soil microbial communities, increased atmospheric CO 2 may impact soil ecosystems indirectly through plant responses. This study tested the hypothesis that exposure of plants to elevated CO 2 would impact soil microorganisms responsible for key nitrogen cycling processes, specifically denitrification and nitrification. We grew trembling aspen (Populus tremuloides) trees in outdoor chambers under ambient (360 ppm) or elevated (720 ppm) levels of CO 2 for 5 years and analyzed the microbial communities in the soils below the trees using quantitative polymerase chain reaction and clone library sequencing targeting the nitrite reductase (nirK) and ammonia monooxygenase (amoA) genes. We observed a more than twofold increase in copy numbers of nirK and a decrease in nirK diversity with CO 2 enrichment, with an increased predominance ofBradyrhizobia-like nirK sequences. We suggest that this dramatic increase in nirK-containing bacteria may have contributed to the significant loss of soil N in the CO 2-treated chambers. Elevated CO 2 also resulted in a significant decrease in copy numbers of bacterial amoA, but no change in archaeal amoA copy numbers. The decrease in abundance of bacterial amoAwas likely a result of the loss of soil N in the CO 2-treated chambers, while the lack of response for archaeal amoA supports the hypothesis that physiological differences in these two groups of ammonia oxidizers may enable them to occupy distinct ecological niches and respond differently to environmental change.

Cytoskeleton, Jun 1, 2021

Proceedings of the National Academy of Sciences of the United States of America, Sep 28, 2020

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2015

Proceedings of the National Academy of Sciences of the United States of America, Mar 3, 2014

Nature Cell Biology, May 25, 2020

Journal of Biological Chemistry, Jul 1, 2011

Current Biology, Dec 1, 2022

Cytoskeleton, 2021

Nature Cell Biology, 2020

Cell-cell fusion is an indispensable process in the conception, development and physiology of mul... more Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamin’s proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically...

Current Biology, 2016

Science (New York, N.Y.), Jan 31, 2015

Developmental Cell, 2015

Proceedings of the National Academy of Sciences, 2014

Nature Structural & Molecular Biology, 2011

Microbial Ecology, 2012