Biology subjects

Calderin, J. D.

Publications and source records attributed to Calderin, J. D..

4 recordsLinked to original sources

bioRxiv2025

Vacuolar Phosphatidylinositol 3,4,5-trisphosphate controls fusion through binding Vam7, and membrane microdomain assembly

Membrane trafficking is regulated by the spatiotemporal distribution of phosphoinositides. The endolysosomal pathway is controlled by PI3P, PI(4,5)P2 and PI(3,5)P2, whereas a role for PI(3,4,5)P3 is less clear. We report that yeast produce PI(3,4,5)P3 through Vps34 activity. In vitro assays showed that dioctanoyl (C8) PI(3,4,5)P3, the PI(3,4,5)P3-binding domain Grp1-PH and the phosphatase PTEN blocked vacuole fusion. Fluorescence microscopy showed that PI(3,4,5)P3 was present at the plasma membrane and vacuoles, and that its detection was blocked by PTEN, C8-PI(3,4,5)P3, the Vps34 inhibitor SAR405 and a VPS34 temperature sensitive mutation. In addition, minimizing PI(4,5)P2 as a substrate for Vps34 with a MSS4 temperature sensitive mutation reduced PI(3,4,5)P3 levels. Importantly, PI(3,4,5)P3 was required for the vertex enrichment of Ypt7 and the HOPS subunit Vps33. Finally, we showed that the soluble SNARE Vam7 was retained in a PI(3,4,5)P3-dependent manner and that its displacement from membranes blocked trans-SNARE pairing. These results demonstrate that vacuolar PI(3,4,5)P3 coordinates vertex assembly and SNARE function. SummarySaccharomyces cerevisiae produces PI(3,4,5)P3 via Vps34 activity on vacuoles to regulate homotypic fusion. PI(3,4,5)P3 functions through controlling vertex microdomain assembly and retaining the soluble SNARE Vam7 on membranes to drive fusion.

biochemistry↗

bioRxiv2025

Broad-Spectrum Activity and Mechanisms of Action of SQ109 on a Variety of Fungi

ABSTRACTWe investigated the activity of the tuberculosis drug SQ109 against sixteen fungal pathogens: Candida albicans, C. auris, C. glabrata, C. guilliermondi, C. kefyr, C. krusei, C. lusitaniae, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, Rhizopus spp., Mucor spp., Fusarium spp., Coccidioides spp., Histoplasma capsulatum and Aspergillus fumigatus. MIC values varied widely (125 ng/mL to >64 {micro}g/mL) but in many cases we found promising (MIC[~]4 {micro}g/mL) activity as well as MFC/MIC ratios of [~]2. SQ109 metabolites were inactive. The activity of 12 analogs of SQ109 against Saccharomyces cerevisiae correlated with protonophore uncoupling activity, suggesting mitochondrial targeting, consistent with the observation that growth inhibition was rescued by agents which inhibit ROS species accumulation. SQ109 disrupted H+/Ca2+ homeostasis in S. cerevisiae vacuoles, and there was synergy (FICI[~]0.31) with pitavastatin, indicating involvement of isoprenoid biosynthesis pathway inhibition. SQ109 is, therefore, a potential antifungal agent with multi-target activity.

microbiology↗

bioRxiv2024

Lysophospholipid headgroup size, and acyl chain length and saturation differentially affect vacuole acidification, Ca2+ transport, and fusion

SNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipids impact fusion through direct protein-lipid interactions or through modulating the physical properties of membranes to affect protein function. Lysophospholipids (LPLs) can affect membrane curvature, fluidity and energy of deformation. Their effects are due to their head group, and the length and saturation of their single acyl chains. Here we examined how the properties of LPLs affect yeast vacuole fusion and ion transport. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons inhibited fusion with IC50 values of {cong} 40-120 {micro}M. While acyl chain length moderately affected fusion, the head group played a major role. Unlike LPCs, Lysophosphatidic acid (LPA 18:1) failed to fully inhibit fusion, while lysophosphatidylethanolamine (LPE 18:1) had no effect. Separately we found that changes in acyl chain length and saturation differentially affected Ca2+ transport and vacuole acidification. Together these data show that the effects of LPLs on membrane fusion and ion transport were due to a combination of head group type and acyl chain length.

biochemistry↗

bioRxiv2022

High throughput analysis of vacuolar acidification

Eukaryotic cells are compartmentalized into membrane-bound organelles, allowing each organelle to maintain the specialized conditions needed for their specific functions. One of the features that change between organelles is luminal pH. In the endocytic and secretory pathways, luminal pH is controlled by isoforms and concentration of the vacuolar-type H+-ATPase (V-ATPase). In the endolysosomal pathway, copies of complete V-ATPase complexes accumulate as membranes mature from early endosomes to late endosomes and lysosomes. Thus, each compartment becomes more acidic as maturation proceeds. Lysosome acidification is essential for the breakdown of macromolecules delivered from endosomes as well as cargo from different autophagic pathways, and dysregulation of this process is linked to various diseases. Thus, it is important to understand the regulation of the V-ATPase. Here we describe a high-throughput method for screening inhibitors/activators of V-ATPase activity using Acridine Orange (AO) as a fluorescent reporter for acidified yeast vacuolar lysosomes. Through this method, the acidification of purified vacuoles can be measured in real-time in half-volume 96-well plates or a larger 384-well format. This not only reduces the cost of expensive low abundance reagents, but it drastically reduces the time needed to measure individual conditions in large volume cuvettes.

biochemistry↗