Biology subjects

Bohannon, K. P.

Publications and source records attributed to Bohannon, K. P..

3 recordsLinked to original sources

bioRxiv2026

Toxoplasma GRA8 engages the host ESCRT accessory protein ALG-2 and is necessary for parasite metabolic integrity

The endosomal sorting complex required for transport (ESCRT) is a hetero-multimeric membrane-remodeling machinery essential for endosomal sorting, intraluminal vesicle formation, cytokinetic abscission, and membrane repair. ESCRT is also hijacked by some pathogens including the protozoan Toxoplasma gondii, which subverts it at the parasitophorous vacuole membrane to support parasite ingestion of host cytosolic proteins. Although the ESCRT accessory protein, ALG-2 is recruited to the parasitophorous vacuole, nothing was known about how this happens. Herein we identify the dense granule protein TgGRA8 as a key effector that recruits ALG-2 and ALIX to the parasitophorous vacuole. We show that TgGRA8 directly binds ALG-2 via conserved ALG-2-binding elements like those found in other ALG-2 interacting proteins including ALIX and SEC31A. Biochemical assays show high-affinity, Ca{superscript 2}-dependent TgGRA8-ALG-2 binding, and structural modeling suggests TgGRA8 may assemble multivalently to coordinate multiple ALG-2 dimers, stabilizing ALG-2/ALIX recruitment through a non-canonical bridging mechanism. Conservation of these motifs among tissue cyst-forming coccidians implies a lineage-linked adaptation that supports infection by these parasites. Metabolomics further indicates that TgGRA8 loss disrupts amino acid, purine, and central carbon metabolism, like those seen in other ingestion deficient mutants. Together, these findings uncover a conserved, multivalent strategy by which Toxoplasma engages host ALG-2 to organize ESCRT at the parasitophorous vacuole, thereby coupling nutrient acquisition to metabolic fitness and exposing a novel agent for probing ESCRT biology.

cell biology↗

bioRxiv2024

Ca2+-sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that widely used concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca2+ release from nearly all. Chelating cytoplasmic Ca2+ using BAPTA makes lysosomes more sensitive to GPN-induced rupture, suggesting that Ca2+ plays a role in protecting or rapidly repairing lysosomal membranes. GPN-elicited Ca2+ release causes the Ca2+-sensor Apoptosis Linked Gene-2 (ALG-2) and ESCRT proteins it interacts with to redistribute onto lysosomes. ALG-2, but not its ESCRT binding-disabled {Delta}GF122 splice variant, improves lysosomal resistance to rupture by GPN or the lysosomotropic drug chlorpromazine. Importantly, directly elevating juxta-lysosomal Ca2+ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca2+, through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress. SignificanceAs the degradative hub of the cell, lysosomes are full of toxic content that can spill into the cytoplasm. There has been much recent interest in how cells sense and repair lysosomal membrane damage using ESCRTs and cholesterol to rapidly fix "nanoscale damage". Here, we extend understanding of how ESCRTs contribute by uncovering a preventative role of the ESCRT machinery. We show that ESCRTs, when recruited by the Ca2+-sensor ALG-2, play a critical role in stabilizing the lysosomal membrane against osmotically-induced rupture. This finding suggests that cells have mechanisms not just for repairing but also for actively protecting lysosomes from stress-induced membrane damage.

cell biology↗

bioRxiv2017

Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on BDNF discharge following fusion

Synuclein is strongly implicated in the pathogenesis of Parkinsons disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of -, {beta}-, and {gamma}-synuclein are comprised of seven 11-amino acid repeats that are predicted to form amphipathic helices. -Synuclein binds to negatively charged lipids, especially small vesicles and tubulates and vesiculates lipids. The membrane-binding and membrane-curving abilities raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous {beta}-synuclein associates with secretory granules. Endogenous -synuclein only rarely is found on secretory granules. Overexpression of -synuclein but not {beta}-synuclein quickened the median duration of the post-fusion discharge of BDNF-pHluorin by 30%, consistent with -synuclein speeding fusion pore expansion.

neuroscience↗