Biology subjects

Hammer, J. A.

Publications and source records attributed to Hammer, J. A..

9 recordsLinked to original sources

bioRxiv2026

A myosin filament cage drives multiple aspects of mitosis unique to cells dividing within a polarized epithelium

Here we used mouse intestinal organoids expressing endogenously tagged myosin to clarify its roles in those aspects of mitosis unique to division within a polarized epithelium. Super-resolution imaging of rounded metaphase cells revealed a never-before-seen myosin filament cage that only forms in this context, exhibits apical/basal polarity, maintains roundness by resisting surrounding tissue pressure, and repositions laterally to form a contractile ring predetermined to drive planar division and asymmetric cleavage furrow progression. Attenuating cage function inhibited interkinetic nuclear migration and metaphase cell rounding, causing the division plane to shift from planar to orthogonal and resulting in organoids possessing multiple lumens. Following the apical domain marker ZO-1 showed that the new lumens originate at the interface between stacked daughter cells created by orthogonal divisions. We conclude that cells dividing within polarized epithelia create a myosin filament cage designed specifically to drive those aspects of cell division unique to their context.

cell biology↗

bioRxiv2025

MYOSIN IIA MOTOR REGULATES ATTACHING-EFFACING BACTERIA INTERACTIONS WITH INTESTINAL EPITHELIUM

Attaching effacing (A/E) bacteria, such as Enteropathogenic E. coli (EPEC) and Citrobacter rodentium, colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via inhibition of pathogen-induced remodeling of the actin cytoskeleton.

cell biology↗

bioRxiv2023

Myosin-5 varies its steps along the irregular F-actin track

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

biophysics↗

bioRxiv2021

A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here we show using super-resolution imaging of mouse primary B cells that LFA-1: ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR): antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation.

immunology↗

bioRxiv2021

Filopodia powered by class X myosin promote fusion of mammalian myoblasts

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. Conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multi-nucleated mammalian muscle formation.

cell biology↗

bioRxiv2020

Endoplasmic reticulum visits highly active spines and prevents runaway potentiation of synapses

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but LTD was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both LTP and LTD are possible.

neuroscience↗

bioRxiv2020

Myosin 18Aα targets guanine nucleotide exchange factor β-Pix to dendritic spines of cerebellar Purkinje neurons to promote spine maturation

Dendritic spines are signaling microcompartments that serve as the primary site of synapse formation in neurons. Actin assembly and myosin 2 contractility play major roles in the maturation of spines from filopodial precursors, as well as in the subsequent, activity-dependent changes in spine morphology that underly learning and memory. Myosin 18A is a myosin 2-like protein conserved from flies to man that lacks motor activity, is sub-stochiometric to myosin 2, and co-assembles with myosin 2 to make mixed filaments. Myosin 18A is alternatively spliced to create multiple isoforms that contain unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. These observations suggest that myosin 18A serves to recruit proteins to mixed filaments of myosin 2 and myosin 18A. One protein known to bind to myosin 18A is {beta}-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42. Notably, {beta}-Pix has been shown to promote spine maturation by activating both Arp2/3 complex-dependent branched actin filament assembly and myosin 2 contractility within spines. Here we show that myosin 18A is expressed in cerebellar Purkinje neurons and concentrates in spines along with myosin 2 and F-actin. Myosin 18A is targeted to spines by co-assembling with myosin 2 and by an actin binding site present in its N-terminal extension. miRNA-mediated knockdown of myosin 18A results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A. Importantly, {beta}-Pix co-localizes with myosin 18A in spines, and its spine localization is lost upon myosin 18A knockdown or when its myosin 18A binding site is deleted. Finally, we show that the spines of myosin 18A knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data demonstrate that mixed filaments of myosin 2 and myosin 18A form a complex with {beta}-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of {beta}-Pixs GEF activity.

neuroscience↗

bioRxiv2019

Characterization of neuronal synaptopodin reveals a myosin V-dependent mechanism of synaptopodin clustering at the post-synaptic sites

The spine apparatus (SA) is an endoplasmic reticulum-related organelle which is present in a subset of dendritic spines in cortical and pyramidal neurons. The synaptopodin protein localizes between the stacks of the spine apparatus and is essential for the formation of this unique organelle. Although several studies have demonstrated the significance of the SA and synaptopodin in calcium homeostasis and plasticity of dendritic spines, it is still unclear what factors contribute to its stability at the synapse and whether the SA is locally formed or it is actively delivered to the spines. In this study we show that synaptopodin clusters are stable at their locations. We found no evidence of active microtubule-based transport for synaptopodin. Instead new clusters were emerging in the spines, which we interpret as the SA being assembled on-site. Furthermore, using super-resolution microscopy we show a tight association of synaptopodin with actin filaments. We identify the actin-based motor proteins myosin V and VI as novel interaction partners of synaptopodin and demonstrate that myosin V is important for the formation and/or maintenance of the SA.

neuroscience↗

bioRxiv2018

Arp2/3 complex-dependent spatial organization of the B cell receptor (BCR) impacts immune synapse formation, BCR signaling and B cell activation

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially-restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodelling in B cell responses to APC-bound antigens.

immunology↗