mDia1 Assembles a Linear F-Actin Coat at Membrane Invaginations To Drive Listeria monocytogenes Cell-to-Cell Spreading

Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion.

The Burkholderia pseudomallei intracellular ‘TRANSITome’

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a ‘TRANSITomic’ approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei ‘TRANSITome’ reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei ‘TRANSITome’ provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.

Switching between blebbing and lamellipodia depends on the degree of Nonmuscle Myosin II activity

Cells can adopt both mesenchymal and amoeboid mode of migration through membrane protrusive activities, namely lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebbing is yet to be explored. Here, we show that addition of ROCK inhibitor, Y27632 or lower doses of (-) blebbistatin, an inhibitor of NMII ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of lower doses of ML7, an inhibitor of MLCK, induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebbing and lamellipodia membrane protrusion are able to maintain pRLC/RLC ratio at cortices when MLCK and ROCK are inhibited, respectively, either pharmacologically or genetically, suggesting that they are interlinked in BLC and LBC. Such BLC and LBC are also inducible in other cells like MCF7 and MCF10A. These studies reveal that relative activity of ROCK and MLCK, which controls both NMII's ATPase activity and filamentous property is a determining factor for a cell to exhibit blebbing or lamellipodia.

Membrane-proximal F-actin restricts local membrane protrusions and directs cell migration

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility

ABSTRACTCell motility is governed by cooperation between the Arp2/3 complex and nucleation factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X-chromosome of multiple animals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-mediated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family proteins.AUTHOR SUMMARYThe actin cytoskeleton is a collection of protein polymers that assemble and disassemble within cells at specific times and locations. Sophisticated cytoskeletal regulators called nucleation factors ensure that actin polymerizes when and where it is needed, and most nucleation factors are members of the Wiskott-Aldrich Syndrome Protein (WASP) family. Several of the 8 known WASP-family proteins function in cell motility, but how the different factors collaborate with one another is not well understood. In this study, we identified WHIMP, a new WASP-family member which is encoded on the X chromosome of a variety of animals. In mouse cells, WHIMP enhances cell motility by assembling actin filaments that push the cell membrane forward. Unexpectedly, WHIMP also activates tyrosine kinase enzymes, proteins that stimulate multiple WASP-family members during motility. Our results open new avenues of research into how nucleation factors cooperate during movement and how the molecular activities that underlie motility differ in distinct cell types and organisms.

Signaling Diversity Enabled by Rap1-Regulated Plasma Membrane ERK with Distinct Temporal Dynamics

A variety of different signals induce specific responses through a common, ERK-dependent kinase cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubtion of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we have expanded the toolbox of FRET-based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK enzymatic activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytopolasm and nucleus. Furthermore, EGF-induced plasma membrane ERK activity involves Rap1, a noncanonical activator, and controls cell morphology and EGF-induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.