Biology subjects

Hundley, F. V.

Publications and source records attributed to Hundley, F. V..

3 recordsLinked to original sources

bioRxiv2024

Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons

Plasma membrane protein degradation and recycling is regulated by the endolysosomal system, wherein endosomes bud from the plasma membrane into the cytosol and mature into degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface, influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinsons to Alzheimers disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we developed a toolkit for capturing EEA1-postive endosomes (Endo-IP) and TMEM192-positive lysosomes (Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrated its utility by revealing the endolysosomal protein landscapes for cortical-like iNeurons and stem cells. This allowed us to globally profile endocytic cargo, identifying hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

cell biology↗

bioRxiv2024

The PRC2.1 Subcomplex Opposes G1 Progression through Regulation of CCND1 and CCND2

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued proliferation inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in antagonizing G1 progression in a diversity of cell linages, including CML, breast cancer and immortalized cell lines.

molecular biology↗

bioRxiv2022

Early Endosome Capture Proteomics and its Application to Amyloid Precursor Protein Intramembrane Processing by β and γ-Secretases

Degradation and recycling of plasma membrane-localized proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. The endolysosomal system also participates in intracellular membrane protein trafficking, signaling, and protein degradation via autophagy. Our understanding of the endolysosomal system has been significantly advanced through the development of methods for selective capture of lysosomes (termed Lyso-IP), but methods for analogous isolation of early intermediates in the endolysosomal system are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of early endosomes in action. In addition to resident endosomal components, we identify numerous recycling, regulatory and membrane fusion complexes as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes that we distinguished from lysosomes via comparative proteomics. The approach allows capture of internalized transferrin within minutes of its addition to cells, indicating the utility of the approach for dynamic analysis of early/sorting endosomes. Among the endocytic cargo identified by Endo-IP was the amyloid precursor protein (APP) genetically linked with Alzheimers disease. Processing of APP to amyloidogenic A{beta} peptides by {beta}- and {gamma}-Secretases can occur within the endolysosomal system among other compartments, but methods for spatial quantification of A{beta} products in individual organelles are lacking. We combined Endo- and Lyso-IP with targeted proteomics to provide a spatial digital snapshot of A{beta} products. This analysis revealed that products of A{beta} processing by {beta}- and {gamma}-Secretases, and alterations in the specificity of cleavage by small molecule {gamma}-Secretase modulators, can be quantified in both early/sorting endosomes and lysosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of the many processes that are coordinated on early endosomes.

cell biology↗