The stochastic nature of DNA damage dictates various scenarios of stress survival among different cells. We used time-lapse microscopy to perform single-cell level analyses of yeast populations with double-stranded DNA breaks and nonfunctional telomeres. Activation of the DNA damage signalling resulted in a broad distribution in the duration of G2/M arrests. Strikingly, the longer arrests correlated with aberrant mitoses caused by miscoordination of nuclear division and cytokinesis, leading to cell death. Chk1-dependent phosphorylation of Pds1 (securin), an inhibitor of sister chromatid separation in mitosis, was responsible for this phenomenon. Securin progressively accumulated during G2/M arrests, leading to grossly delayed or missed nuclear divisions. This phenotype could be suppressed by slowing down the progression of mitotic exit via LTE1 deletion or by lowering securin levels. Our results demonstrate that in cells taking too long to complete DNA repair, the DNA damage checkpoint promotes aberrant mitoses and cell death, thereby eliminating cells with a higher chance of genomic instability.

The human ciliopathy Alström syndrome is caused by mutations in ALMS1. How ALMS1 contributes to cilia function has been unclear. Unlike other ciliopathy-associated proteins, ALMS1 localizes to the proximal end of centrioles, raising the possibility that it contributes to cilia function via a distinct mechanism. We found that, without ALMS1, centrioles are short and frequently break at their proximal ends. Truncated, broken ALMS1 mutant centrioles display reduced microtubule-organizing activity and disrupted centriole linkers. Without ALMS1, Golgi-derived cargos, including transmembrane components of the transition zone, are impaired in their trafficking to the centrosome. Consequently, ALMS1 is important for transition zone assembly, ciliogenesis, and the control of ciliary membrane composition. In mice, disruption of ALMS1 causes progressive, age-dependent loss of cilia in tissues affected in Alström syndrome, including retina, kidney, and brain. We conclude that Alström syndrome is caused by disruption of proximal centriole integrity and centrosome-directed protein trafficking, compromising ciliary assembly and maintenance.

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 Saccharomyces cerevisiae produces PI(3,4,5)P3 through Vps34 activity using PI(4,5)P2 as a substrate. 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 on vacuoles and the plasma membrane and that its presence was eliminated by inhibiting Vps34. Importantly, PI(3,4,5)P3 was required for the enrichment of Ypt7 and the HOPS subunit Vps33 at vertex domains. Blocking PI(3,4,5)P3 also caused the release of the soluble SNARE Vam7, leading to inhibited trans-SNARE pairing. Finally, we show that PI(3,4,5)P3 is needed for vacuole acidification in vivo. These results demonstrate that PI(3,4,5)P3 affects multiple factors in vacuole function.

Clathrin-mediated endocytosis is a conserved eukaryotic trafficking process where an Arp2/3 complex-nucleated branched actin network provides force for vesicle formation. The mechanisms that initiate endocytic actin assembly are incompletely understood. In the fission yeast, Schizosaccharomyces pombe, actin assembly is initiated by Dip1, an Arp2/3 complex activator. In the budding yeast, Saccharomyces cerevisiae, the initiation of actin assembly has remained a mystery. Here, we show that S. cerevisiae Ldb17, the homolog of Dip1, functions as an on-switch for endocytic actin assembly. Unexpectedly, the regulation of Ldb17 is more complicated than that of constitutively active Dip1. Ldb17 is controlled by a coat protein, Sla1, via separate recruitment and activation steps. This regulation was likely lost in the S. pombe lineage, and this simplification may be related to other changes in actin assembly between these species. Our findings add a key missing piece in the understanding of endocytosis in S. cerevisiae and reveal an intriguing evolutionary tinkering of the actin on-switch.

In budding yeast, Lte1, a component of the spindle position checkpoint, must be confined to the bud for correctly coupling chromosomal segregation across the bud neck with mitotic exit. Bud compartmentalization involves a phospho-dependent program with Ras-GTP acting as the ultimate cortical anchor. Here, we have dissected this program by identifying an Lte1 domain participating in Ras binding and two phospho-regulatory motifs, the N- and C-Lte1 regulatory elements (LREs). The C-LRE mediates control of Lte1 cortical recruitment by the p21-activated kinase, Cla4. The N-terminal LRE modulates Lte1 binding to Ras more directly and is phosphorylated by the yeast MARK/PAR-1 protein kinases, Kin1/2. Along with previously described cyclin-dependent kinase phosphorylation sites, the N- and C-LREs are required for Lte1 localization and timely mitotic exit. These studies uncover the coordinated interplay between multiple protein kinases, allowing Lte1 to integrate cell polarity and cell cycle cues for strict compartmentalization at the bud cell cortex.

Export of secretory proteins at ER exit sites (ERES) has been described to occur via COPII-coated vesicles, tubules, or tunnels. These models have not been considered to reflect the flexibility of ERES but rather as mutually exclusive mechanisms. Here, we overview these export mechanisms, discuss why they coexist, and hypothesize about the biological relevance of carrier geometry. Finally, we suggest experimental avenues to resolve the disagreement.

Recent work from two independent studies has uncovered mitochondria as a previously unrecognized proton source driving lysosomal acidification. In this Viewpoint, we summarize these findings, examine the associated proteins and functional mechanisms, and discuss their relevance to disease.

Chromosome segregation is mediated by microtubule-based machinery, the spindle. Microtubule-independent structures, often called the spindle matrix or the spindle envelope, have been reported to embed or surround the spindle, respectively. The concepts have been around for several decades, but they still remain enigmatic. Here, we show the spindle matrix and the spindle envelope form one functional unit crucial for spindle structure and function in oocytes. We found that in Drosophila oocytes, Slender lobes (Sle) protein is an integral component of the spindle matrix, which has a composition different from that in mitotic cells. We further found that Greatwall (Gwl) kinase localizes to the spindle envelope tightly surrounding the Sle spindle matrix in oocytes. Gwl promotes assembly of the Sle spindle matrix, which in turn determines the location of the Gwl spindle envelope. sle silencing in oocytes resulted in spindle abnormalities. Cosilencing experiments revealed that Sle buffers the impacts of compromised kinetochore-microtubule interaction on centromere bi-orientation in meiotic metaphase I.

Animal cytokinesis is often described as constriction of a RhoA-dependent actomyosin contractile ring. Yet, this ring operates within a shrinking membrane-cortex interface into which cortical flow continually delivers mechanically engaged material, creating a mass-balance problem: how can the interface close without becoming congested? Here, I propose a membrane-centric framework in which sustained furrow ingression depends on coordinated Loading and Relief. Actomyosin contractility gathers and compresses membrane-cortex material at the furrow apex, while membrane-proximal, RhoA-dependent anillo-septin assemblies are proposed to facilitate slip-mediated Lateral Relief by routing material away from the contractile ring proper. The ring remains central, but within a wider three-dimensional cleavage zone where cortical lamination allows inflow and outflow to occur simultaneously. This framework predicts that furrow progression and robustness depend on efficient redistribution of membrane-cortex material, while geometrical advance need not directly report material-processing rate. It suggests direct tests of whether impaired disengagement and exit cause material retention, altered furrow dynamics, and congestion.

The cytoskeleton is a key mediator of mechanical interactions in cells, but specific contributions of septins remain unclear. Septins preferentially localize with a subset of actin stress fibers positioned under the nucleus, where they are situated between the membrane and stress fibers. Removing the nucleus from the cell results in the loss of these subnuclear septin-decorated stress fibers. Surprisingly, however, their formation can be rescued using a large glass bead in place of the nucleus. Similarly, applying a compressive force to the cell via confinement, whether externally or through internally generated actomyosin forces, results in increased septin accumulation in regions where the nucleus engages the cell cortex. Finally, loss of septin filaments via knockdown of SEPT7 increases nuclear membrane rupture during confinement. Together, these data argue that septins act as a dynamic mechanosensitive protective network to buffer mechanical forces on the nucleus.

Invasively migrating cells thread their nucleus through confined interstitial spaces. How cells protect the nucleus from intracellular forces is poorly understood. Here, we show that the septin cytoskeleton buffers the actomyosin forces that have been shown to power nuclear movement. Septin filaments comprising SEPT9, a septin amplified in breast cancer, align with perinuclear actomyosin cables, which exhibit higher tensile stress during 3D confined migration through narrower pores. SEPT9 depletion amplifies actin stress during confined migration and after myosin II hyperactivation in nonmigrating cells causing actin and nuclear envelope ruptures. Following confined migration, DNA breaks, nuclear blebs, micronuclei, and cell death increase in SEPT9-depleted cells, phenotypes rescued by the oncogenic SEPT9 isoform 1. Clinicogenomic data reveal that SEPT9 amplification associates with lower genomic alteration in aggressive breast tumors. We propose that septins are a mechanoprotective element of the cytoskeleton, and SEPT9 amplification enhances tumor cell survival by preventing nuclear damage.

E-cadherin suppresses invasion and promotes metastasis in invasive ductal breast cancer, through suppression of reactive oxygen species (ROS). We sought to determine the cellular source of apoptosis-inducing ROS and to identify the domains of E-cadherin that are essential for cancer cell survival. Inhibition of mitochondria-derived ROS was sufficient to rescue cell death following E-cadherin deletion, in both 3D colony formation and in vivo metastasis assays. We next generated Cdh1 constructs by mutating amino acids or deleting domains, then tested their ability to rescue colony formation in E-cadherin-deleted cells. Surprisingly, E-cadherin constructs in which the extracellular domains were deleted, adhesion was compromised, or catenin interactions were blocked could rescue elevated mitochondrial ROS and 3D colony formation. Conversely, a construct lacking the cytoplasmic domain was unable to rescue ROS or cell death. Our findings reveal that E-cadherin's ability to regulate mitochondrial ROS, cancer cell survival, and metastatic outgrowth is separable from its role in intercellular adhesion.

Weakening of adherens junctions (AJs) and focal adhesions (FAs) adhesiveness has been proposed to enable cancer initiation and progression. Here we report that E-cadherin at cell-cell contacts and vinculin at FAs are transiently under higher tension in MCF10A cells overactivating the Src proto-oncogene. Knockdown of paxillin or P-cadherin, or inhibition of focal adhesion kinase (FAK) or AJ assembly with EGTA, suppresses this force increase across both E-cadherin and vinculin. FAK and P-cadherin trans-homophilic interactions further enhance Src-dependent myosin II and extracellular signal-regulated kinase (ERK) activation. In addition, FAK and P-cadherin potentiate ERK and serum response factor (SRF) signaling, thereby supporting cell proliferation. Furthermore, knockdown of Drosophila cadherin or alpha integrin suppresses Src-induced tissue overgrowth, reinforcing the role of adhesion integrity in Src-driven proliferation. Thus, prior to the loss of cell-cell and cell-matrix adhesiveness, adhesion integrity by P-cadherin at AJs and FAK at FAs could represent a critical step enabling premalignant cells to gain a proliferative advantage.

Robust chromosome segregation requires the anaphase spindle to both preserve and remodel its structure under force. How it does so remains unclear as probing mechanics during anaphase's short lifetime is challenging. Here, we use microneedles to pull on mammalian anaphase midzone bundles and ask how they respond to and transmit force across space and time. We find that midzone bundles locally transmit force to each other in the spindle's short axis over multiple timescales. Along the spindle's long axis, midzone bundles globally transmit force: rather than bundles sliding apart or detaching under force, the spindle shortens. This reveals strong anchorage and resistance to outward sliding, and that spindle elongation requires all midzone bundles to elongate. This global force transmission is stronger for short-lived forces and is strictly PRC1-dependent, indicating limited mechanistic redundancy. In sum, the anaphase spindle acts as a single mechanical unit over short timescales to resist and transmit force, while remodeling over long timescales to segregate chromosomes.