For decades, the social amoeba Dictyostelium discoideum has been an invaluable tool for dissecting the biology of eukaryotic cells. Its short growth cycle and genetic tractability make it ideal for a variety of biochemical, cell biological, and biophysical assays. Dictyostelium have been widely used as a model of eukaryotic cell motility because the signaling and mechanical networks which they use to steer and produce forward motion are highly conserved. Because these migration networks consist of hundreds of interconnected proteins, perturbing individual molecules can have subtle effects or alter cell morphology and signaling in major unpredictable ways. Therefore, to fully understand this network, we must be able to quantitatively assess the consequences of abrupt modifications. This ability will allow us better control cell migration, which is critical for development and disease, in vivo. Here, we review recent advances in imaging, synthetic biology, and computational analysis which enable researchers to tune the activity of individual molecules in single living cells and precisely measure the effects on cellular motility and signaling. We also provide practical advice and resources to assist in applying these approaches in Dictyostelium.
Cell division requires constriction of an actomyosin ring to segregate the genetic material equally into two daughter cells. The spatial and temporal regulation of the contractile ring at the division plane primarily depends on intracellular signals mediated by the centralspindlin complex and astral microtubules. Although much investigative work has elucidated intracellular factors and mechanisms controlling this process, the extracellular regulation of cytokinesis remains unclear. Thus far, the extracellular matrix protein Hemicentin (HIM-4) has been proposed to be required for cleavage furrow stabilization. The underlying molecular mechanism, however, has remained largely unknown. Here, we show that HIM-4 and anillin (ANI-1) genetically act in the same pathway to maintain the rachis bridge stability in the germline. Our FRAP experiments further reveal that HIM-4 restricts the motility of ANI-1. In addition, we demonstrate that HIM-4 is recruited to the cleavage site in dividing germ cells and promotes the proper ingression of the cleavage membrane. Collectively, we propose that HIM-4 is an extracellular factor that regulates ANI-1 for germ cell membrane stabilization and contractile ring formation in Caenorhabditis elegans germline cells.
Functions of Ras oncogenes and their downstream effectors are typically associated with cell proliferation and growth control while their role in immune cell migration has been largely unexplored. Although Ras-mediated signaling cascades have been implicated in immune response, there is no conclusive evidence to show local activation of these pathways on the plasma membrane directly regulates cell motility or polarity. Using spatiotemporally precise, cryptochrome-based optogenetic systems in human neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant-sensing receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or the guanine nucleotide exchange factor, RasGRP4, immediately increased spreading and random motility in neutrophils. Second, creating Ras activity at the cell rear generated new protrusions at the site and reversed pre-existing polarity, similar to the effects of steep chemoattractant gradients. Third, recruiting GTPase activating protein, RASAL3, at cell fronts abrogated existing protrusions and changed the direction of motility whereas dynamically inhibiting nascent fronts stopped migration completely. Fourth, combining pharmacological inhibition studies with optogenetics revealed that mTorC2 is more important than PI3K for Ras-mediated polarity and migration. Finally, local recruitment of Ras-mTorC2 effector, Akt, also generated new protrusions, rearranged pre-existing polarity, and triggered migration, even in absence of PI3K signaling. We propose that actin assembly, cell shape, and migration modes in immune cells are promptly controlled by rapid, local activities of established components of classical growth-control pathways independently of receptor activation.
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