Biology subjects

Broadbent, D.

Publications and source records attributed to Broadbent, D..

4 recordsLinked to original sources

bioRxiv2022

Systematic Analysis of Autophagy Identifies Atg9 Vesicles as the Origin of the Phagophore

Autophagy is a catabolic pathway required for the clearance and recycling of cytoplasmic materials. Upregulation and dysfunction of autophagy contributes to the pathology of cancer and neurogenerative diseases, respectively. To define the molecular mechanisms that control autophagic flux it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of 9 cell lines expressing HaloTagged autophagy factors from their endogenous loci, we systematically analyze the abundance, single-molecule dynamics, and autophagosome association kinetics of a wide variety of autophagy proteins involved in the initiation and maturation of the autophagosome. Our results reveal that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles and tethering of these ATG9 vesicles to donor membranes by ATG2 is a key step in phagophore maturation. In addition, we demonstrate that the overall lifetime of an autophagosome is approximately 160 seconds and the majority of phagophore initiation events fail to produce mature autophagosomes. In total our work establishes a new experimental framework to quantitatively analyze autophagy and demonstrates that ATG9 vesicles are the seeds for autophagosome formation in human cells.

cell biology↗

bioRxiv2022

Systematic analysis of the molecular and biophysical properties of key DNA damage response factors

Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling. Genomic insertion of the HaloTag at the endogenous loci of the repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to DSBs, and defined the diffusion dynamics and chromatin binding by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end joining, does not exist in a preassembled state and Shieldin components are recruited to DSBs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by the PST repeat domain of MDC1. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

cell biology↗

bioRxiv2021

In vitro and in vivo analysis of microvesicle-mediated metastasis using a bright, red-shifted bioluminescent reporter protein of extracellular vesicles

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. Our study focused on a novel method to image EV subtypes and their biodistribution in vivo. Regardless of injection routes, we established that reporter EVs isolated from murine mammary carcinoma cells expressing PalmReNL, which utilizes bioluminescence resonance energy transfer (BRET), localized to the lungs. This new EV reporter allowed highly sensitive EV tracking in vitro and in vivo and enabled us to begin studies to understand the commonalities and functional differences of the EV subtypes. We demonstrated the early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple injections of the microvesicle (MV)-enriched fraction derived from mammary carcinoma cells. In addition, the results we present here show that tumor cell-derived MVs act on distant tissues through upregulating LC3 expression within the lung.

cancer biology↗

bioRxiv2020

A Single-Objective Light-Sheet Microscope with 200 nm-Scale Resolution.

We present an Oblique Plane Microscope that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of Lattice Light-Sheet Microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

biophysics↗