The nuclear pore complex (NPC) mediates nucleocytoplasmic transport while contributing to nuclear organization and genome regulation. In Saccharomyces cerevisiae , the nucleoporin Nup170, positioned in the NPC's inner ring, plays a critical role in subtelomeric gene silencing, telomere tethering to the nuclear envelope, and association of silencing factors with subtelomeric chromatin. However, the molecular basis of these functions and their relationship to NPC architecture remain unclear. Here, we define a nucleoplasm-exposed region of Nup170 required for subtelomeric gene silencing. Systematic truncation analysis reveals that Nup170 residues between ∼990 and 1200 are necessary to restore silencing, telomere positioning, and subtelomeric gene expression in nup170 Δ cells. Mapping these truncations onto high-resolution structural models of the NPC places this region on a nucleoplasmic-facing surface of the inner ring, accessible through openings in the outer ring scaffold. Targeted single amino acid substitutions within this region, including nup170 D1099G , phenocopy the subtelomeric silencing defect, telomere tethering defect, and reduced SIR complex binding of nup170 Δ. These results demonstrate that a short, structurally defined surface of Nup170 within the NPC scaffold directly supports epigenetic regulation at the nuclear periphery. Our findings reveal how architectural nucleoporins perform specific genome regulatory functions through discrete interfaces, highlighting functional specialization among individual NPCs.
The deregulation of neural stem cell (NSC) homeostasis underlies neurodevelopmental disorders and tumorigenesis. In the Drosophila larval brain, NSCs divide asymmetrically along an invariant apical-basal axis to yield a larger self-renewing stem cell and a smaller, differentiating sibling. NSC divisions are instructed by centrosomes, which recruit pericentriolar material (PCM) important for microtubule-organizing activity. During interphase, NSC centrosomes are defined by asymmetries in PCM composition and functional activity. These asymmetries help align the mitotic spindle and position the cleavage furrow. Here, we show that the conserved RNA-binding protein Orb2 is required to enrich Polo kinase, the master regulator of centrosome maturation, at centrosomes throughout the cell cycle. Loss of Polo correlates with compromised localization of downstream PCM effectors in orb2 NSCs. Despite these impairments, orb2 NSCs build and orient their spindles normally to complete asymmetric cell division. However, through live imaging and clonal analysis, we demonstrate that Orb2 is required to maintain proper cell size, as loss of orb2 yields significantly smaller NSCs and sibling cells. We further show that Orb2 supports the timely clearance of cortical myosin important for cleavage plane positioning. These data indicate that Orb2 promotes NSC centrosome maturation and size of the neural progenitors.
Endosomal Sorting Complexes Required for Transport (ESCRT) machinery drives membrane remodeling and scission events essential for multivesicular body (MVB) biogenesis. The accessory ESCRT-III subunit Vps60 (mammalian CHMP5) shares structural features with the major polymerizing subunit Snf7, including the capacity to form homopolymers in vitro . Here, we demonstrate in Saccharomyces cerevisiae that Vps60 recruitment and polymerization require conditions that permit Snf7 polymer turnover: Vps60 is largely excluded when Snf7 assembly is absent and when Snf7 disassembly is blocked, consistent with recruitment of Vps60 late in the ESCRT-III cycle to Snf7 assemblies that undergo normal turnover. Structure-function analysis reveals that Vps60 regulation diverges fundamentally from core ESCRT-III subunits: while occlusion of the Vps60 C terminus by a GFP tag drives constitutive membrane association and polymer assembly, C-terminal truncation impairs endosomal recruitment, indicating that the Vps60 C terminus is required for membrane recruitment, in contrast to the autoinhibitory C-terminal domains of core ESCRT-III subunits. A C-terminally tagged Vps60 that constitutively associates with membranes phenocopies loss of Vps60 function in both intralumenal vesicle (ILV) formation and cargo sorting. These findings indicate that despite structural similarity to Snf7, Vps60 functions as a late-arriving accessory factor whose recruitment and assembly are tightly coupled to Snf7 dynamics.
The TORC1 (Target of Rapamycin Complex 1) kinase complex coordinates eukaryotic cell growth and metabolism in response to nutrient availability. In yeast, one of the proteins under TORC1 control is the Npr1 kinase mediating nitrogen control of several plasma membrane transporters, yet the metabolic signals governing this pathway remain poorly understood. Here, we first investigated the previously described and unexpected activation of TORC1-Npr1 signaling upon acute glucose starvation. We show that this response occurs only under specific nitrogen conditions, notably in cells grown on proline or glutamine, and depends on increased ATP production driven by stimulated respiration. In proline-grown cells, this respiration is fueled by catabolism of α-ketoglutarate, while high α-ketoglutarate levels negatively affect TORC1-Npr1 activation. We next analyzed TORC1-Npr1 activation following NH 4 + addition to proline-grown cells. Our results confirm that glutamine accumulation is not required for this activation and instead show that NH 4 + assimilation is associated with a decrease in α-ketoglutarate levels and a sustained increase in ATP production, concomitant with TORC1-Npr1 activation. In cells lacking the NH 4 + -assimilating glutamate dehydrogenases, both increased ATP production and TORC1-Npr1 activation are impaired. Together, these findings identify ATP and α-ketoglutarate as important metabolic determinants of TORC1-Npr1 signaling in response to nitrogen availability.
While lysosomes are typically globular in morphology, lipopolysaccharide-activated macrophages reorganize lysosomes into an expanded tubular network. Here, we sought to determine if the V-ATPase and the lysosomal pH contribute to LPS-mediated lysosome tubulation in macrophages. We found that inhibition of the V-ATPase prevented lysosome tubulation and collapsed preformed tubules. However, the V-ATPase also controls mTORC1 activity, which itself is needed for tubulation. To distinguish between lysosomal pH and mTORC1, we turned to NH 4 Cl, which alkalinized lysosomal pH but did not interfere with mTORC1; yet NH 4 Cl blocked tubulation showing that an acidic lysosomal pH is needed for lysosome remodelling. Moreover, clamping the pH to either acidic or alkaline pH caused tubules to collapse, indicating that the pH gradient promotes tubulation, rather than a specific pH. These effects were not due to altered microtubule organization or impaired lysosome motility, suggesting that motors remained associated with lysosomes. On the other hand, while LPS did not alter the average pH of spherical or tubular lysosomes, growing tubules displayed a more acidic peripheral end relative to the pericentral end. Based on this observation, we propose that a localized pH gradient along the tubule may enable tubulation by modulating factors that catalyse tubule growth.
