RESUMO
During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).
RESUMO
Actin assemblies are important in motile cells such as leukocytes, which form dynamic plasma membrane extensions or podia. L-plastin (LCP1) is a leukocyte-specific calcium-dependent actin-bundling protein that, in mammals, is known to affect immune cell migration. Previously, we generated CRISPR/Cas9 engineered zebrafish lacking L-plastin (lcp1-/-) and reported that they had reduced survival to adulthood, suggesting that lack of this actin-bundler might negatively affect the immune system. To test this hypothesis, we examined the distribution and migration of neutrophils and macrophages in the transparent tail of early zebrafish larvae using cell-specific markers and an established wound-migration assay. Knockout larvae were similar to their heterozygous siblings in having equal body sizes and comparable numbers of neutrophils in caudal hematopoietic tissue at 2 days postfertilization, indicating no gross defect in neutrophil production or developmental migration. When stimulated by a tail wound, all genotypes of neutrophils were equally migratory in a two-hour window. However, for macrophages we observed both migration defects and morphological differences. L-plastin knockout macrophages (lcp1 -/-) still homed to wounds but were slower, less directional and had a star-like morphology with many leading and trailing projections. In contrast, heterozygous macrophages lcp1 (+/-) were faster, more directional, and had a streamlined, slug-like morphology. Overall, these findings show that in larval zebrafish L-plastin knockout primarily affects the macrophage response with possible consequences for organismal immunity. Consistent with our observations, we propose a model in which cytoplasmic L-plastin negatively regulates macrophage integrin adhesion by holding these transmembrane heterodimers in a "clasped," inactive form and is a necessary part of establishing macrophage polarity during chemokine-induced motility.