Biology subjects

Millard, S. S.

Publications and source records attributed to Millard, S. S..

5 recordsLinked to original sources

bioRxiv2026

Single-molecule bouncing at a liquid interface governs presynaptic enrichment

Many cellular processes rely on accumulating highly diffusive cytoplasmic proteins to a specific location within the cell. This spatial enrichment is generally thought to rely on locally immobilizing proteins or slowing diffusion through binding and molecular crowding. Paradoxically, cytoplasmic proteins that are enriched within nerve terminals remain highly diffusive, raising questions about the mechanisms that govern presynaptic enrichment. Here, using in vivo single-molecule microscopy, we tracked the diffusion of a model cytoplasmic protein, -Synuclein (-Syn), and synaptic vesicles (SVs), in motor neuron presynapses of live Drosophila larvae. We demonstrate that -Syn undergoes rapid and frequent "bouncing" events at a presynaptic interface, locally impeding escape from the presynapse. We measured escape probabilities at the interface, and show that impeding escape, without dramatically slowing diffusion, is sufficient to govern presynaptic enrichment. The interface is liquid-like and coincides with the boundary of the SV cluster, which is a necessary scaffold for the presynaptic enrichment of -Syn. Our results provide direct evidence of interfacial reflectivity at the interface of liquid-like compartments, and that this phenomenon can govern the subcellular enrichment of proteins and small organelles at the level of individual particles.

neuroscience↗

bioRxiv2024

The microcephaly protein WDR62 regulates cellular purine metabolism through the HSP70/HSP90 chaperone machinery.

Inherited mutations in WD repeat-containing protein 62 (WDR62) are associated with microcephaly (MCPH2). While WDR62 plays important roles in mitosis and centriole biogenesis, additional WDR62 functions may cause abnormal brain growth. Here, we reveal a novel WDR62 role in the molecular chaperone network regulating purine metabolism. In response to hyperosmotic stress, WDR62 redistributes to purinosomes--phase-separated membraneless assemblies of purine metabolic enzymes and their chaperones. While WDR62 is not needed for purinosome formation, its loss disrupts purine homeostasis, resulting in the accumulation of purine nucleotide intermediates and a reduction in the levels of hypoxanthine-guanine phosphoribosyl transferase (HPRT), a key purine salvage enzyme. We link this to WDR62s interaction with Bcl2-associated athanogene 2 (BAG2), a co-chaperone that modulates the function of HSP70/90. In cells lacking WDR62, BAG2 levels are elevated and HPRT stability is reduced. Knocking down BAG2 in these cells restores HPRT levels, underscoring the crucial role of WDR62-BAG2 interactions in chaperone-mediated stability and turnover of metabolic pathway enzymes. Notably, common microcephaly-associated mutations in WDR62 alter its interaction with BAG2, suggesting that purine metabolic defects resulting from WDR62 mutations may underlie microcephaly in humans.

cell biology↗

bioRxiv2024

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both basal and induced mitophagy. Disruption of the phosphatase activity of PPTC7 is not required for BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-NIX/BNIP3 and PPTC7-FBXL4 interactions and their disruption interferes with both NIX/BNIP3 degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts NIX/BNIP3-induced mitophagy.

cell biology↗

bioRxiv2023

Regulated alternative splicing of Dscam2 is required for somatosensory circuit wiring

Axon and dendrite placement and connectivity is guided by a wide range of secreted and surface molecules in the developing nervous system. Nevertheless, the extraordinary complexity of connections in the brain requires that this repertoire be further diversified to precisely and uniquely regulate cell-cell interactions. One important mechanism for molecular diversification is alternative splicing. Drosophila Down syndrome cell adhesion molecule (Dscam2) undergoes cell type-specific alternative splicing to produce two isoform-specific homophilic binding proteins. Regulated alternative splicing of Dscam2 is important for dendrite and axon patterning, but how this translates to circuit wiring and animal behavior is not well understood. Here, we examined the role of cell-type specific expression of Dscam2 isoforms in regulating synaptic partner selection in the larval somatosensory system. We found that synaptic partners in the nociceptive circuit express different Dscam2 isoforms. Forcing synaptic partners to express a common isoform resulted in nociceptive axon patterning defects and attenuated nocifensive behaviors, indicating that a role for Dscam2 alternative splicing is to ensure that synaptic partners do not express matching isoforms. These results point to a model in which regulated alternative splicing of Dscam2 across populations of neurons restricts connectivity to specific partners and prevents inappropriate synaptic connections.

neuroscience↗

bioRxiv2018

Muscleblind regulates Drosophila Dscam2 cell-type-specific alternative splicing

Alternative splicing of genes increases the number of distinct proteins in a cell. In the brain it is highly prevalent, presumably because proteome diversity is crucial for establishing the complex circuitry between trillions of neurons. To provide individual cells with different repertoires of protein isoforms, however, this process must be regulated. Previously, we found that the mutually exclusive alternative splicing of a cell surface protein, Dscam2 produces two isoforms (exon 10A and 10B) with unique binding properties. This splicing event is tightly regulated and crucial for maintaining axon terminal size, dendritic morphology and synaptic numbers. Here, we show that Drosophila Muscleblind (Mbl), a conserved splicing factor implicated in myotonic dystrophy, controls Dscam2 alternative splicing. Removing mbl from cells that normally express isoform B induces the expression of isoform A and eliminates the expression of B, demonstrating that Mbl represses one alternative exon and selects the other. Mbl mutants exhibit phenotypes that are also observed in flies engineered to express a single isoform. Consistent with these observations, mbl expression is cell-type-specific and correlates with the expression of isoform B. Our study demonstrates how the regulated expression of a splicing factor is sufficient to provide neurons with unique protein isoforms crucial for development.

neuroscience↗