Molecular Biology of the Cell

The actin fold is present in enzymes ranging from sugar kinases to chaperones. Previous work on the actin fold metabolic enzyme, glucokinase (Glk1p) in S. cerevisiae found it could form filaments in response to its substrates, ATP and glucose (Stoddard et al., 2020). Here, we have identified the product, glucose 6-phosphate (G6P), as a second trigger for Glk1p polymerization in vitro. Furthermore, the addition of ADP to G6P-Glk1p filaments causes filament disassembly, suggesting that polymerization is sensitive to the state of the bound nucleotide and/or the transfer of the gamma phosphate. We have also identified a specific metabolic state, the accumulation of G6P during stationary phase, that triggers Glk1p polymerization in vivo. While the structures of Glk1p filaments in either the ATP/glucose or G6P-bound form are not similar to conventional actin filaments, the sensitivity of assembly to the gamma phosphate of the nucleotide provides a conceptual bridge between the cytoskeleton and metabolic regulation via enzyme polymerization.

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

Proper cell physiology requires the co-assembly of multiple actin cytoskeletal networks that are tailored for specific functions. To maintain and promote the different functions of these networks, cells decorate them with distinct types of actin binding proteins (ABPs). While various models have been proposed to explain this selective sorting of ABPs, the role of actin disassembly factors is less well understood. Here, we used inducible CRISPR interference and quantitative live-cell imaging to test how disassembly factors control the ABP composition of different networks. We found that knockdown of cofilin (Cof1), a potent and highly conserved disassembly factor, disrupts the size, organization, and ABP composition of actin networks. Specifically, defects in Cof1-mediated disassembly disrupt intracellular transport due to the assembly of overgrown and disordered branched actin networks that are inappropriately decorated by tropomyosin (Tpm1). Contrary to prevailing models of ABP sorting, these networks are co-decorated by Tpm1 and fimbrin (Sac6), and their assembly is independent of formin activity. Instead, our findings support a model wherein failure to maintain the proper architecture of branched actin networks drives mis-localization of network-specific ABPs. Together, this work demonstrates that actin disassembly factors play a critical role in maintaining cytoskeletal structure and function to regulate ABP sorting across distinct networks.

While bioelectricity is increasingly recognized as an important regulator of cell function and morphogenesis, the field has almost exclusively focused on plasma membrane states. Voltage across the inner nuclear membrane (INM) has been proposed as a potential regulator of nuclear function, but how it responds to extracellular ionic perturbations and whether it relates to chromatin organization remain unclear. Here, we targeted the ratiometric genetically encoded voltage indicator ASAP3-R3 to SUN2-associated nuclear membranes in intact NRK cells and combined INM voltage measurements with Gray-Level Co-Occurrence Matrix (GLCM)-based chromatin texture analysis. Reporter localization was confirmed by fluorescence imaging and electron microscopy, and functional validation in isolated nuclei showed that sodium-potassium pump inhibition produced INM depolarization consistent with Goldman-Hodgkin-Katz (GHK)-based prediction. We then used our validated construct to determine the response of Vnuc and chromatin texture to changing ionic conditions via two exposure methods, gradual (ramped) exposure or direct application. In intact cells, ramping different sets of ionic solutions of decreasing sodium/increasing potassium, decreasing sodium, increasing potassium, or decreasing chloride induced INM hyperpolarization and coordinated changes in chromatin texture, including increased contrast and entropy, reduced homogeneity, and reduced nuclear area. These effects were strongly path-dependent, with nuclear responses shaped by the history and order of ionic exposure: sodium and potassium responses emerged most clearly during ramping exposure, whereas reducing chloride by direct exposure showed a more pronounced response profile. Direct changes in sodium exposure produced limited electrical and chromatin-texture effects, while direct potassium exposure altered chromatin texture and nuclear area without significantly changing VNuc. Importantly, shifting baseline chromatin state in either direction, through Trichostatin-A (TSA)-induced chromatin relaxation or sodium azide/2-deoxy-D-glucose-induced compaction, blunted ion-associated Vnuc and chromatin responses across sodium, potassium, and chloride conditions. Together, these findings identify the nucleus as a dynamic, ion-responsive electro-structural system in which INM voltage and chromatin organization are functionally coupled, and in which both ionic trajectory and pre-existing chromatin state shape the magnitude of the nuclear response.

Polyploid cells, which contain more than two copies of the genome, are widely present across plants and animals, where they are often found in tissues with high biosynthetic and metabolic demands, such as the mammalian liver and placenta. While somatic polyploidy is frequently associated with increased cell growth and biosynthetic capacity, unscheduled polyploidization in cell types that are not normally programmed to become polyploid is often linked to reduced cellular fitness and genome instability. To understand whether these divergent outcomes stem from distinct immediate cellular responses to increased ploidy, we systematically compared the early consequences of polyploidization across naturally occurring and experimentally induced systems. Specifically, we examined physiological polyploid cells in the Caenorhabditis elegans intestine and human hepatocyte organoids, alongside unscheduled polyploid human retinal pigment epithelial (RPE1) cells generated through cytokinesis failure. Using quantitative imaging, flow cytometry, and FUCCI based cell-cycle reporters we measured cell size and protein translation dynamics during G1 in diploid and polyploid cells. Across all systems, we observed a strikingly conserved relationship between ploidy, cell size, and biosynthetic capacity: both cell size and protein translation showed similar scaling patterns after polyploidization, regardless of whether polyploidization occurred as part of normal development or by inducing cytokinesis failure. These findings indicate that the immediate cellular response to increased ploidy is broadly similar across contexts. However, in contrast to unscheduled polyploid RPE1 cells, polyploid human hepatocytes extend their G1 phase, leading to a higher accumulation of proteins before cell-cycle progression. Together, our findings suggest that polyploidization elicits similar growth responses across contexts, and that cell-type specific cell-cycle adaptations may determine whether polyploidy becomes advantageous or deleterious.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

BackgroundThe fission yeast stress-activated protein kinase (SAPK) pathway includes a conserved mitogen-activated protein (MAP) kinase cascade that regulates multiple cellular processes and is activated by several types of external stress. Understanding how Sty1, the MAP kinase in the SAPK pathway, controls these processes is complicated by the fact that different stressors can have stressor-specific effects that may be difficult to separate from the effects of Sty1 activation itself. Moreover, upon stress, Sty1 activation is usually short-lived. Previously, we developed a fission yeast strain, SISA, in which Sty1 kinase activity can be switched on in a sustained manner in the absence of external stress. This required combining multiple mutations in the SAPK pathway, including an analog-sensitive version of Sty1. When SISA cells are grown in the presence of analog-sensitive kinase inhibitors, Sty1 is inhibited, but when inhibitor is removed, Sty1 becomes hyperactive. While this strain was useful, it had several limitations. ResultsHere we describe and validate a more rationally-designed strain, SISA4, that retains the features of the original SISA strain while overcoming its limitations. SISA4 is more stable genetically than SISA, easier to use in genetic crosses, and easy to identify by phenotype or genotyping. We show that analog-sensitive kinase inhibitors 4-Amino-1-tert-butyl-3-(1-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1) and 4-Amino-1-tert-butyl-3-(3-bromobenzyl)pyrazolo[3,4-d]pyrimidine (3-BrB-PP1) are equally potent for inhibiting analog-sensitive Sty1 in vivo, and we determine optimal inhibitor concentrations for converting SISA4 cells from a Sty1-inhibited state to a Sty1-hyperactive state. We also find that both 1-NM-PP1 and 3-BrB-PP1 have measurable off-target effects in wild-type cells, although these are modest and generally do not affect interpretation of experiments. Finally, using SISA4, we show that the Sty1-activated transcription factor Atf1 plays an unexpected role in maintaining cell-polarity disruption after Sty1 hyperactivation. ConclusionsSISA4 will be useful for investigating how SAPK pathway activation regulates diverse cellular processes.

Saturated cultures of the pathogenic yeast, Cryptococcus neoformans, arrest as unbudded cells in the G2 phase of the cell cycle. As cells divided and cultures saturated, we found that oxygen levels in the culture medium dropped nearly tenfold. When saturation-arrested cultures were re-oxygenated without adding fresh growth medium, cells immediately formed a bud and then underwent mitosis. Thus, the arrest is due to low oxygen concentration rather than nutrient depletion. Because the G2 arrest was associated with unbudded cells, we asked whether C. neoformans cells have a morphogenesis checkpoint that blocks mitosis until cells can form a bud. Inhibition of budding by treatment with Latrunculin A also led to G2 arrest, and we determined that this arrest is dependent on the CDK inhibitory kinase, Swe1. This finding suggests that C. neoformans possesses a morphogenesis checkpoint analogous to that in the distantly related Saccharomyces cerevisiae. We also demonstrated that Swe1 is required to enforce the hypoxia-induced G2 arrest. We propose that hypoxia inhibits budding in C. neoformans, which in turn triggers a morphogenesis checkpoint to arrest cells in G2 even when nutrients are plentiful.

Rab GTPase cascades drive endosomal membrane maturation by sequentially activating and inactivating Rab proteins. These transitions in Rab signaling require the coordinated actions of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The yeast VINE complex is an endosomal VPS9-family GEF that stimulates a GAP to inactivate the Rab5 homolog Vps21, suggesting a role for VINE in coordinating Rab transitions. Here we report that VINE acts through its catalytic GEF domain to promote signaling by the Rab5-related GTPase Ypt10 and establish a pool of Ypt10 at late endosomes. Ypt10 activation occurs downstream of Vps21 activity, placing Ypt10 within a late endosomal Rab cascade. Genome-wide protein proximity screens revealed a VINE-dependent interaction between Ypt10 and the GEF Mon1-Ccz1. Our data suggest that VINE and Ypt10 regulate late endosomal recruitment of Mon1-Ccz1 to enhance the activation of its substrate, the Rab7 homolog Ypt7. Together, these findings define a Vps21-VINE-Ypt10 regulatory module that adds a layer of control within the late endosomal Vps21-to-Ypt7 cascade and establish VINE as a dual Rab regulator. Through opposing activities on Vps21 and Ypt10, VINE may couple Rab5 inactivation to Mon1-Ccz1 recruitment to provide more precise control of degradative protein traffic to the vacuole. Significance statementFour Rab5-family GTPases direct protein sorting and membrane maturation in the yeast endolysosomal system, yet their individual functions, and the role of the little-studied Rab Ypt10, are unclear. Using genome-wide proximity screens, we find that the GEF complex VINE establishes a pool of Ypt10 at late endosomes downstream of Vps21, where Ypt10 recruits Mon1-Ccz1, the activator of the Rab7 homolog Ypt7. Because VINE also drives GAP-mediated suppression of Vps21, our results suggest it acts as a dual Rab regulator, coupling Vps21 inactivation to Ypt10 activation to fine-tune the endosomal Rab cascade.

Mucolipidosis type IV is a lysosomal storage disease that is characterized by delayed psychomotor development and retinal degeneration due to cell death, in addition to other symptoms that are due to aberrant functions of live tissues. Caenorhabditis elegans CUP-5 is the orthologue of human TRPML1, the protein that is dysfunctional in Mucolipidosis type IV patients. Mirroring Mucolipidosis type IV pathology, loss of C. elegans CUP-5 results in developing intestinal cell death in embryos leading to embryonic lethality, while other tissues in adults lacking CUP-5 are alive but dysfunctional. We had previously shown that ESCRT-Associated proteins and the ATP-Binding Cassette Transporter MRP-4 are necessary for acquiring aberrant and poorly functional lysosomes in the absence of CUP-5. In this study, we show that the aberrant lysosomes permeabilize or rupture, thus releasing lysosomal degradative enzymes that kill cells in the absence of CUP-5. We also show that the autophagy-related protein ATG-9 mediates, in an autophagy-independent manner, this lysosomal permeabilization. We finally propose phenotypic and biochemical models linking CUP-5 to lysosomal defects and cell death.

Entry of intracellular pathogenic bacteria is widely considered an actin-driven process, potentially overlooking contributions of the microtubule cytoskeleton. Here, we identify a host microtubule state as a determinant of early infection efficiency by Chlamydia pneumoniae. Human cells enriched in acetylated microtubules are preferentially infected, whereas detyrosinated microtubules show no such association. Pharmacological stabilization of microtubules via Taxol enhances infection, while selective elevation of acetylation with Tubacin does not, indicating that microtubule stability rather than acetylation alone is critical. Thus, a pre-existing interphase microtubule architecture supports C. pneumoniae entry. Consistently, mitotic cells, characterized by a reorganized microtubule architecture, remain permissive but show severely reduced infection efficiency. In addition, infection induces a dose-dependent increase in microtubule acetylation that requires bacterial viability and is not observed during uptake of Yersinia pseudotuberculosis effector protein Invasin-coated beads, indicating that entry/internalization alone is insufficient to trigger this response. To probe how early chlamydial secreted effectors might engage the microtubule cytoskeleton, we focused on the conserved TarP family member CPn0572, an actin and microtubule regulator, which increases microtubule acetylation when ectopically expressed in human cells. Controlled expression of microtubule-localized CPn0572 in the yeast Schizosaccharomyces pombe leads to altered microtubule dynamic and mechanical behaviour, promoting force-bearing microtubules. Together, these findings show that distinct interphase microtubules define a permissive cellular state for bacterial entry and suggest that early chlamydial effector activities might promote a specific microtubule persistence phenotype.

Cell wound repair requires rapid and coordinated remodeling of the actin cytoskeleton to restore cortex integrity. Here, we show that a Rab35-Mical-SelR pathway regulates actomyosin ring dynamics through reversible actin redox. We find that Rab35 is recruited to wounds and is essential for proper actin ring assembly and disassembly. Rab35 regulates the recruitment of Mical, an actin-oxidizing enzyme, and SelR, a reductase that reverses oxidation, to the cell wound. Mical and SelR knockdowns disrupt actin ring formation and wound closure, whereas double knockdown partially rescues these defects, indicating a balanced redox cycle is required. Super-resolution microscopy reveals that Mical and SelR differentially regulate F-actin architecture and orientation. Mutation of actin at Methionine 44 does not fully recapitulate Mical knockdown phenotypes, suggesting the presence of additional targets and enzymes. Taken together, our results indicate that reversible actin modifications dynamically regulate F-actin architecture and orientation for actin ring assembly and disassembly.

Centrosomes organize microtubules, coordinate ciliogenesis, and support accurate cell division. At the mature mother centriole, distal and subdistal appendages confer specialized functions in ciliary docking, microtubule organization, and intracellular trafficking. Nuclear distribution element 1 (NDE1) is a centrosome-associated regulator of dynein-dependent processes and ciliogenesis, but its nanoscale organization and contribution to centrosome architecture remain incompletely understood. Here, using expansion microscopy and STED super-resolution imaging, we show that endogenous NDE1 forms a ring-like structure at the subdistal appendages in mouse embryonic fibroblasts and human RPE-1 cells. NDE1 occupies an intermediate radial position between the more centriole-proximal CEP128 layer and the more peripheral ninein layer. Depletion of ODF2 or CEP128 reduces centrosomal NDE1, whereas CEP170 depletion has little effect, placing NDE1 within an ODF2- and CEP128-dependent branch of the subdistal appendage organization network. NDE1 depletion compromises centrosome integrity, reduces the centrosomal enrichment of core centriolar proteins, increases the separation between paired centrioles, and generates ectopic foci containing multiple centriolar markers. Loss of NDE1 also disrupts pericentriolar material organization and impairs the establishment of focused, centrosome-associated microtubule arrays. Furthermore, NDE1 depletion increases LC3B- and p62-positive structures and reduces autophagic flux. Together, our findings establish NDE1 as a subdistal appendage-associated factor that supports centrosome architecture and microtubule-organizing activity. More broadly, they support an emerging view of subdistal appendages as a molecularly layered platform in which distinct but cooperating components connect mother centriole maturation to microtubule organization, ciliary regulation, and intracellular trafficking.

Lysosomes are critical for neuronal physiology and synaptic function, but their organization and roles within astrocytes, an integral component of the tripartite synapse, remain unknown. Here, we use a neuron-astrocyte coculture system that promotes stellate astrocyte morphology to investigate the trafficking of late endosomes and lysosomes (LELs) in astrocytes. As astrocyte branches mature, degradative activity becomes concentrated in the soma and LELs in branches undergo bidirectional motility that becomes progressively attenuated. We establish that the lysosomal cation channel TRPML1 drives LEL immobilization. TRPML1-mediated arrest involves myosin-Va tethering to the actin cytoskeleton, which may position LELs near peripheral astrocyte processes (PAPs), fine actin-enriched protrusions that can contact synapses. Strikingly, TRPML1 activity modulates the phosphorylation of ezrin, radixin, and moesin, actin-membrane linkers enriched in PAPs. These effects are rapid and transient, suggesting a role for lysosomal TRPML1 in regulating PAP membrane dynamics. Thus, TRPML1 positions LELs proximal to PAPs which may influence PAP structural plasticity and astrocyte-synapse contacts.

Chromosome segregation errors in oocyte meiosis are a leading cause of early miscarriage and congenital disorders in mammals and these errors become more prevalent with advanced maternal age. Although the effects of aging on the functions of critical meiotic proteins and cytoskeletal filaments in oocytes are known, the influence of aging on the force generating capabilities of oocyte spindle components remains largely unexplored. Through the integration of a coarse-grained model and in situ experiments, we compare the long-axis mechanical properties of metaphase I (MI) and II (MII) oocyte spindles from reproductively young and old mice. Increased inter-kinetochore distance in aged MII oocytes agree with a model of age-associated cohesion loss, and kinetochore dynamics in these spindles following laser ablation suggest a similar reduction in inter-kinetochore bridge viscosity. Simultaneously, we find that both cohesive and poleward force generators lose stiffness with advanced age in MI spindles. In total, we quantify the extent to which structural spindle components lose their stiffness and viscosity during maternal aging, highlighting the multifaceted impacts of aging on mouse oocyte spindle mechanics. Significance StatementO_LIMaternal aging influences mammalian oocyte spindles in numerous ways, yet the impacts of aging on the balance of collective spindle forces remain poorly understood. C_LIO_LIIntegrating coarse-grained mechanical modeling with in situ measurements of spindle morphology and kinetochore dynamics, we quantify age-associated changes to the viscosities and elastic stiffnesses of oocyte spindle component parts. C_LIO_LIThis work provides both a characterization of the effects of aging on force production in mammalian oocyte spindles and a blueprint for future studies of spindle force generation in complex biological contexts. C_LI

The eukaryotic cytoskeleton generates remarkable diversity in cellular architecture despite being built from deeply conserved actin and tubulin polymers. Diversification of cytoskeletal regulators, motors, and filament-organizing proteins produces highly varied cellular morphologies across eukaryotes, yet certain higher-order architectures repeatedly emerge in distantly related lineages. One notable example is cytoskeletal arborization, which occurs not only in metazoan neurons but also in amoeboid lineages distributed throughout the eukaryotic tree. Whether these similar branched architectures arise through conserved cytoskeletal organization, independent reuse of shared molecular systems, or convergence driven by common physical constraints remains unresolved. Here, we investigate the rhizarian amoeba Filoreta ramosa, which forms a multinucleate reticulated network through branching and anastomosis. Using live imaging, immunofluorescence, morphometric analyses, and cytoskeletal drugs, we define how actin and microtubule systems organize branch formation, intracellular transport, and large-scale network architecture. Actin-rich protrusions initiate exploratory branchlets that become selectively stabilized through microtubule incorporation. Longitudinal microtubule arrays reinforce mature branches and support rapid bidirectional organelle transport, while branch nodes function as distributed sites of microtubule nucleation. These cytoskeletal features parallel key mechanisms underlying neuronal arborization, including actin-driven exploration, microtubule-dependent branch stabilization, and transport systems that scale with increasingly extended cytoskeletal networks. However, unlike neurons, Filoreta develops a decentralized reticulated network through repeated anastomosis and distributed microtubule organization, demonstrating that similar arborized morphologies can emerge through distinct architectural strategies. Our findings indicate that arborization can arise through multiple evolutionary adaptations to common cellular constraints. Shared cytoskeletal mechanisms repeatedly support branching architectures, but distinct topologies and modes of cellular organization demonstrate that evolution can reach arborization through different routes. Similar cytoskeletal networks may repeatedly emerge in diverse lineages when cells face the challenges of exploration, stabilization, and transport across increasingly larger scales. Filoreta therefore provides an experimentally tractable model for investigating how conserved cytoskeletal systems generate diverse arborized cellular architectures across eukaryotic evolution.

Dsn1 and Ndc80 are essential proteins of the kinetochore complex and required for chromosome segregation in dividing cells and for regulating development and microtubule dynamics in postmitotic neurons. With conditional deletion of floxed alleles, we here show that Dsn1 and Ndc80 are also required for the viability of postmitotic neurons, both in cultures of hippocampal and cortical neurons and in vivo in the retina. Loss of these proteins triggers apoptosis, as indicated by caspase cleavage and an increase in nuclear DNA breakage. The pro-survival function of the kinetochore components is distinct from that which was previously demonstrated for regulation of neuronal synaptogenesis. The microtubule-binding domain of Ndc80 is required for the synaptogenic functions but Ndc80 lacking this domain can nonetheless rescue the viability of neurons from which Ndc80 has been deleted. Similarly, whereas the synaptogenic function involves regulation of microtubules in axons and dendrites, a nucleus-localized Dsn1 is sufficient to rescue the viability of neurons from which Dsn1 has been deleted. Thus, postmitotic neurons retain a nuclear requirement for components of the kinetochore in order to prevent apoptosis.

The polarization of motile cilia requires that the centrioles, from which cilia are formed, display rotational asymmetry. This property is manifested in the presence of asymmetrically distributed appendages and relies on evolutionary conserved mechanisms. These mechanisms are also at play in cells that form primary cilia despite the lack of ciliary motility and asymmetric centriole appendages in this context. Here, we find that a complex consisting of CCDC61, KIAA1328 (K1328), and Centlein (CNTLN) contributes to the establishment of centriole rotational asymmetry. In cells with a primary cilium, this complex is required for assembling a linker that repositions the daughter centriole close to and orthogonal to the proximal end of the mother centriole/basal body. The CCDC61/K1328/CNTLN complex also triggers the asymmetric recruitment of pericentriolar matrix components around newly assembled centrioles, which likely facilitates the later attachment of the basal body-daughter centriole linker. Overall, our results establish that rotational asymmetry relies on the coordinated recruitment of asymmetric landmarks along centrioles and is necessary for positioning the centrioles in a configuration that is widely conserved in ciliated cells.

The Arp2/3 complex has long been considered to only assemble branched actin structures in the cell (lamellipodia, endocytic patches, comet tails, and many more). We show for the first time by single-molecule tracking (SMT) that the Arp2/3 complex and SPIN90, which activates Arp2/3 complex to nucleate unbranched filaments, bind to and move in the basal cortex with stress fibers and focal adhesions (FA) that, unlike known sites of Arp2/3 enrichment, employ linear actin bundles. SPIN90 knockout in U2OS cells significantly increases the rate of collective cell migration while decreasing cellular traction (myosin-II and actin speeds) and adhesion (FA size and maturation markers). SPIN90's SH3 domain, similar to its adapter protein Nck1, shows enrichment in FAs, suggesting a possible location for SPIN90-Arp2/3 complex activity. Together, our findings indicate that SPIN90-Arp2/3 nucleated filaments also function in stress fibers where they help define the mechanics of traction and adhesion to regulate cell motility.

Quantitative image analysis of subcellular organization requires sufficient spatial resolution to resolve individual organelles and sample size to capture heterogeneity both within cells and between cells. Existing imaging approaches often force a tradeoff between spatial resolution and throughput, limiting the ability to measure organelle-level phenotypes across cell populations. Here, we establish high-throughputs expansion microscopy (HiExM) as a scalable pipeline for single-organelle analysis. As a benchmark, we focus on mapping late endosomes and lysosomes (LELs), a heterogeneous organelle class whose small size, dense intracellular distribution, and functional diversity make it difficult to quantify accurately using conventional light microscopy. HiExM increases effective spatial resolution while preserving compatibility with large-scale image acquisition, enabling robust segmentation and quantitative profiling of individual LELs across large cell populations. Using this pipeline, we identified differences in intracellular trafficking behavior among anti-transferrin receptor antibodies that could not be captured by conventional colocalization analysis alone. We further integrate spatial and morphological features with learned image-based representations that can define relationships between LEL morphology and subcellular position as well as how these relationships respond to perturbations. Together, our work establishes HiExM as a generalizable platform for scalable single-organelle profiling, enabling an analytical framework for quantifying discrete organelles across cells and conditions.