Satarupa Das

mTORC2 Regulates Neutrophil Chemotaxis in a cAMP- and RhoA-Dependent Fashion

We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA-signaling axis, independently of actin reorganization during neutrophil chemotaxis.

Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

RasC controls the spatial and temporal activity of TORC2 to regulate directional cell migration.

Asymmetric nanotopography biases cytoskeletal dynamics and promotes unidirectional cell guidance

Significance Directed motion of cells is essential to many natural and physiological processes, including development and wound healing. Although gradients in chemical or physical properties are well known to guide cells, gradients that are strong enough to guide cells can only cover limited distances. Here we demonstrate that materials with local asymmetries in nano/microtopography on subcellular scales are able to guide cells with a single preferred direction. Unlike conventional gradients, local asymmetries on subcellular scales can be repeated over arbitrarily large regions, providing directional guidance over an unlimited range. This unidirectional guidance is mediated by biasing of the direction of waves of polymerization of the cell’s actin scaffolding, a mechanism that is conserved from primitive cells to human cells. Many biological and physiological processes depend upon directed migration of cells, which is typically mediated by chemical or physical gradients or by signal relay. Here we show that cells can be guided in a single preferred direction based solely on local asymmetries in nano/microtopography on subcellular scales. These asymmetries can be repeated, and thereby provide directional guidance, over arbitrarily large areas. The direction and strength of the guidance is sensitive to the details of the nano/microtopography, suggesting that this phenomenon plays a context-dependent role in vivo. We demonstrate that appropriate asymmetric nano/microtopography can unidirectionally bias internal actin polymerization waves and that cells move with the same preferred direction as these waves. This phenomenon is observed both for the pseudopod-dominated migration of the amoeboid Dictyostelium discoideum and for the lamellipod-driven migration of human neutrophils. The conservation of this mechanism across cell types and the asymmetric shape of many natural scaffolds suggest that actin-wave-based guidance is important in biology and physiology.

Real-time measurements of cAMP production in live Dictyostelium cells.

Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP sensor that possesses high cAMP sensitivity and great temporal resolution. We show the transient profile of cAMP accumulation in live Dictyostelium cells and establish that chemoattractants control intracellular cAMP dynamics by regulating synthesis via the adenylyl cyclase ACA. aca– cells show no significant change in FRET response following chemoattractant addition. Furthermore, cells lacking ACB, the other adenylyl cyclase expressed in chemotaxing cells, behave similarly to wild-type cells. We also establish that the RegA is the major phosphodiesterase that degrades intracellular cAMP in chemotaxis-competent cells. Interestingly, we failed to measure intracellular cAMP compartmentalization in actively chemotaxing cells. We conclude that cytosolic cAMP, which is destined to activate PKA, is regulated by ACA and RegA and does not compartmentalize during chemotaxis.

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G protein-coupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.

Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.

Cell Motility and Nanolithography

Abstract In this chapter, we review how two-photon polymerization may be utilized to systematically fabricate nanotopographies that mimic the environment of a cell, for example, in our body. These nanofabricated topographies allow for systematic studies of a broad range of important biological processes that are affected by the texture of the surroundings of a cell.

Adenylyl cyclase mRNA localizes to the posterior of polarized DICTYOSTELIUM cells during chemotaxis

BackgroundIn Dictyostelium discoideum, vesicular transport of the adenylyl cyclase A (ACA) to the posterior of polarized cells is essential to relay exogenous 3′,5′-cyclic adenosine monophosphate (cAMP) signals during chemotaxis and for the collective migration of cells in head-to-tail arrangements called streams.ResultsUsing fluorescence in situ hybridization (FISH), we discovered that the ACA mRNA is asymmetrically distributed at the posterior of polarized cells. Using both standard estimators and Monte Carlo simulation methods, we found that the ACA mRNA enrichment depends on the position of the cell within a stream, with the posterior localization of ACA mRNA being strongest for cells at the end of a stream. By monitoring the recovery of ACA-YFP after cycloheximide (CHX) treatment, we observed that ACA mRNA and newly synthesized ACA-YFP first emerge as fluorescent punctae that later accumulate to the posterior of cells. We also found that the ACA mRNA localization requires 3′ ACA cis-acting elements.ConclusionsTogether, our findings suggest that the asymmetric distribution of ACA mRNA allows the local translation and accumulation of ACA protein at the posterior of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signal during chemotaxis.

Back localized adenylyl cyclase a mRNA is actively translated in live chemotaxing Dictyostelium cells

ABSTRACT Dictyostelium discoideum cells transport adenylyl cyclase A (ACA)-containing vesicles to the back of polarized cells to relay exogenous cAMP signals during chemotaxis. Fluorescence in situ hybridization (FISH) experiments showed that ACA mRNA is also asymmetrically distributed at the back of polarized cells. By using the MS2 bacteriophage system, we now visualize the distribution of ACA mRNA in live chemotaxing cells. We found that the ACA mRNA localization is not dependent on the translation of the protein product and requires multiple cis-acting elements within the ACA-coding sequence. We show that ACA mRNA is associated with actively translating ribosomes and is transported along microtubules towards the back of cells. By monitoring the recovery of ACA–YFP after photobleaching, we observed that local translation of ACA–YFP occurs at the back of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signals during chemotaxis. Summary: The relay of cAMP signals is essential for streaming during Dictyostelium chemotaxis. We report the dynamic enrichment and translation of the mRNA encoding adenylyl cyclase A at the back of cells during this process.