Biology subjects

Stabley, D. R.

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

3 recordsLinked to original sources

bioRxiv2023

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen- Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

cell biology↗

bioRxiv2022

Antagonistic action of Siah2 and Pard3/JamC to promote germinal zone exit of differentiated cerebellar granule neurons by modulating Ntn1 signaling via Dcc

Germinal zone (GZ) exit is at the top of a cascade of events promoting the maturation of neurons and their assembly into neuronal circuits. Developing neurons and their progenitors must interpret varied niche signals like morphogens, guidance molecules, extracellular matrix, or adhesive cues to navigate GZ occupancy. How newborn neurons integrate multiple cell-extrinsic niche cues with their cell-intrinsic machinery in exiting a GZ is unknown. We establish cooperation between cell polarity-regulated adhesion and Netrin-1 signaling comprises a coincidence detection circuit repelling maturing neurons from their GZ. In this circuit, the Partitioning defective 3 (Pard3) polarity protein and Junctional adhesion molecule-C (JAM-C) adhesion protein promote, while the Seven in absentia 2 (Siah2) ubiquitin ligase inhibits, Deleted in colorectal cancer (DCC) receptor surface recruitment to gate differentiation linked repulsion to GZ Netrin-1. These results demonstrate cell polarity as a central integrator of adhesive- and guidance cues cooperating to spur GZ exit. Highlights-Netrin1 expressed in the cerebellar GZ ranges from an attractive to repulsive cue depending on differentiation status or extracellular matrix context. -Modulation of DCC expression levels impacts the nature of the Netrin1 response and GZ occupancy status. -Siah2, Pard3, and JamC function to modulate responsiveness to purified Netrin1 and cooperate with Dcc to regulate GZ exit. -Pard3 and JamC promote both basal and Netrin1 stimulated DCC surface recruitment to control GZ exit and Netrin1 repulsion

neuroscience↗

bioRxiv2019

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

cell biology↗