Biology subjects

Vale, R. D.

Publications and source records attributed to Vale, R. D..

22 records · Page 2Linked to original sources

bioRxiv2017

Cellular reconstitution of apoptotic cell clearance reveals a multi-step phosphorylation mechanism for Draper receptor triggering

The engulfment of apoptotic cells is essential for tissue homeostasis and responding to damage. Engulfment is mediated by receptors that recognize ligands exposed on apoptotic cells, such as phosphatidylserine (PS). Here, we convert Drosophila S2 cells into proficient phagocytes by transfecting the Draper engulfment receptor and replacing apoptotic cells with PS-coated beads. We show that PS-ligated Draper forms microclusters that exclude a bulky transmembrane phosphatase and recruit phosphotyrosine binding proteins, revealing a triggering mechanism similar to the T cell receptor (TCR). Analogous to the TCR, Drapers extracellular domain and PS can be replaced with FRB and FKBP respectively, resulting in a rapamycin-inducible engulfment system. However, in contrast to the TCR, we show that localized signaling at Draper microclusters results in time-dependent depolymerization of actin filaments. Collectively, our results reveal mechanistic similarities and differences between the receptors involved in apoptotic corpse clearance and mammalian immunity and demonstrate that engulfment can be reprogrammed towards non-native targets.\n\nCondensed titleSpatial Control of Engulfment Signaling

cell biology↗

bioRxiv2017

The actin cytoskeleton governs apical mitosis and daughter cell dispersion in intestinal epithelia

Cell proliferation is critical for maintaining the absorptive, protective and regenerative functions of the small intestine throughout adulthood. Interphase nuclei are positioned near the basal surface of the intestinal epithelium, but during mitosis, chromosomes are located apically. The molecular basis for apical-basal DNA positioning and its consequences for tissue homeostasis are poorly understood. Here, we image and pharmacologically perturb these behaviors in live murine intestinal organoids. We find that apical and basal DNA movements occur as a result of mitosis-coupled actin rearrangements that alter the basolateral shape of dividing cells, while the apical cell surface remains confined by cell-cell contacts that persist throughout mitosis. Strikingly, these polarized shape changes allow neighboring cells to insert between nascent daughters, intermingling cells of different lineages. In summary, polarized rearrangements of the actin cytoskeleton govern the mitotic behavior of intestinal epithelial cells and lead to interspersion of cell lineages.

cell biology↗

bioRxiv2017

RNA Gelation in Repeat Expansion Disorders

Expansions of short nucleotide repeats in the protein coding and non-coding regions of >30 genes produce a variety of neurological and neuromuscular disorders including Huntingtons disease (CAG repeats), muscular dystrophy (CTG repeats) and amyotrophic lateral sclerosis (GGGGCC repeats) [1-3]. Expression of expanded repeats alone is sufficient to recapitulate disease pathology in animal models [4-6]. Repeat-containing transcripts accumulate in the nucleus as aberrant \"RNA foci\" [7-10] and sequester numerous RNA binding proteins [11,12], leading to a disruption of cellular homeostasis [13,14]. Interestingly, RNA foci, as well as the disease symptoms, only manifest at a critical threshold of nucleotide repeats: >30 for CAG/CTG expansions [1] and >7 for the GGGGCC expansion [15]. However, the reason for this characteristic threshold, as well as the molecular mechanism of foci formation, remain unresolved [16]. Here, we show that nucleotide repeat expansions in RNA create templates for multivalent Watson-Crick (CAG/CUG expansions) or Hoogsteen (GGGGCC expansion) base-pairing. These multivalent interactions cause purified RNAs containing repeat expansions to undergo a sol-gel transition and form micron-sized clusters. Reflecting an increase in the valency for intermolecular hybridization, the gelation of purified RNA only occurs above a critical number of trinucleotide or hexanucleotide repeats. These thresholds for in vitro RNA gelation are similar to those associated with manifestation of disease. By visualizing RNA in live cells, we show that nuclear foci form as a result of phase separation of the repeat-containing RNA and that these foci can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

biophysics↗

bioRxiv2016

T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition

Programmed death-1 (PD-1) is a co-inhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). Here, by titrating the strength of PD-1 signaling in both biochemical reconstitution systems and in T cells, we demonstrate that the coreceptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1- recruited Shp2 phosphatase. We also show that PD-1 colocalizes with the costimulatory receptor CD28 in plasma membrane microclusters but partially segregates from the TCR. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways may play unexpected roles in regulating effector T cell function and therapeutic responses to anti-PD-L1/PD-1.

biochemistry↗