Biology subjects

Linehan, J. B.

Publications and source records attributed to Linehan, J. B..

3 recordsLinked to original sources

bioRxiv2022

Trajectory classification method for anchored molecular motor-biopolymer interactions in the C. elegans first mitosis.

During zygotic mitosis, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, proper segregation of genetic material, and successful cell division. Identification of individual cortical force generating units in vivo is necessary to study the regulation of microtubule dependent force generation throughout the cell cycle, to further understanding of asymmetric cell division, and to identify the molecular mechanism of force generation. Here we present a method to determine both the location and relative number of microtubule dependent cortical force generating units using single molecule imaging of fluorescently labelled dynein. Dynein behavior is modeled to differentiate and categorize trajectories that correspond to that which is cortically bound and interacting with a microtubule, and is cortically bound and not interacting with a microtubule. The categorization strategy recapitulates well known force asymmetries in the first mitosis of the C. elegans embryo. To evaluate the robustness of categorization, we RNAi depleted the microtubule subunit TBA-2 resulting in reduction of the number of trajectories categorized as engaged with a microtubule. This technique will be a valuable tool to provide new insight to the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (eg. Actin, tubulin, DNA).

cell biology↗

bioRxiv2022

Follow that cell: leukocyte migration in L-plastin mutant zebrafish.

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 L-plastin 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 two days post-fertilization, 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 still homed to wounds but were slower, less directional and had a star-like morphology with many leading and trailing projections. In contrast, wild type macrophages were faster, more directional, and had a more 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.

developmental biology↗

bioRxiv2021

Single-particle tracking of dynein identifies PP2A B55/SUR-6 as a cell cycle regulator of cortical force generation

Convergence and positioning of the pronuclei and mitotic spindle of many zygotes aids efficient division and is essential for early embryonic patterning. In the C. elegans zygote, interactions between microtubules and cortically anchored dynein are key to early development. However, how cortical microtubule pulling forces are controlled through the cell cycle is less well understood. We used single-molecule imaging and a windowed mean squared displacement analysis to uncover the behavior of dynein during cortical force generation, and provide a regulatory role for protein phosphatase PP2A-B55/SUR6 via NuMA-like protein LIN-5 in this process. Previous findings and our results suggest that PP2A regulates cortical microtubule pulling forces by increasing dynein binding and unbinding to the cortical force generation complex. Our data also suggests that cortical occupancy of dynein is abrogated to vary force generation. Our approach will be broadly applicable to classify the force generation behavior of single molecules in living organisms.

cell biology↗