Precise measurement of nanoscopic septin ring structures in deep learning-assisted quantitative superresolution microscopy

The combination of image analysis and fluorescence superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning-based object recognition enables the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, deep learning methods are prone to different forms of bias. A biased recognition of structures may prohibit the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. What is required to overcome this problem is a detailed investigation of potential sources of bias and the rigorous testing of trained models using real or simulated data covering a wide dynamic range of possible results. Here we combine single molecule localization-based superresolution microscopy of septin ring structures with the training of several different deep learning models for a quantitative investigation of bias resulting from different training approaches and finally quantitative changes in septin ring structures. We find that trade-off exists between measurement accuracy and the dynamic range of recognized phenotypes. Using our trained models, we furthermore find that septin ring size can be explained by the number of subunits they are assembled from alone. Our work provides a new experimental system for the investigation of septin polymerization.

ARP-T1-associated Bazex–Dupré–Christol syndrome is an inherited basal cell cancer with ciliary defects characteristic of ciliopathies

Actin-Related Protein-Testis1 (ARP-T1)/ACTRT1 gene mutations cause the Bazex-Dupré-Christol Syndrome (BDCS) characterized by follicular atrophoderma, hypotrichosis, and basal cell cancer. Here, we report an ARP-T1 interactome (PXD016557) that includes proteins involved in ciliogenesis, endosomal recycling, and septin ring formation. In agreement, ARP-T1 localizes to the midbody during cytokinesis and the basal body of primary cilia in interphase. Tissue samples from ARP-T1-associated BDCS patients have reduced ciliary length. The severity of the shortened cilia significantly correlates with the ARP-T1 levels, which was further validated by ACTRT1 knockdown in culture cells. Thus, we propose that ARP-T1 participates in the regulation of cilia length and that ARP-T1-associated BDCS is a case of skin cancer with ciliopathy characteristics.

Turgor-dependent and coronin-mediated F-actin dynamics drive septin disc-to-ring remodeling in the blast fungus

The fungus Magnaporthe oryzae uses a specialized pressure-generating infection cell called an appressorium to break into rice leaves and initiate disease. Appressorium functionality is dependent on the formation of a cortical septin ring during its morphogenesis, but precisely how this structure assembles is unclear. Here we show that F-actin rings are recruited to the circumference of incipient septin disc-like structures in a pressure-dependent manner, and that this is necessary for their contraction and remodeling into rings. We demonstrate that the structural integrity of these incipient septin discs requires both an intact F-actin and microtubule cytoskeleton and provide fundamental new insight into their functional organization within the appressorium. Lastly, using proximity-dependent, labelling we identify the actin modulator coronin as a septin proximal protein and show that F-actin-mediated septin disc-to-ring remodeling is perturbed in the genetic absence of coronin. Taken together, our findings provide new insight into the dynamic remodeling of infection-specific higher-order septin structures in a globally significant fungal plant pathogen.

Rsr1 Palmitoylation and GTPase Activity Status Differentially Coordinate Nuclear, Septin, and Vacuole Dynamics in Candida albicans

ABSTRACT Directional growth and tissue invasion by hyphae of the pathogenic fungus, Candida albicans, are disrupted by deletion of the small GTPase, Rsr1, which localizes Cdc42 and its kinase, Cla4, to the site of polarized growth. We investigated additional abnormalities observed in rsr1Δ hyphae, including vacuole development, cytoplasm inheritance, mitochondrial morphology, septin ring organization, nuclear division and migration, and branching frequency, which together demonstrate a fundamental role for Rsr1 in cellular organization. Rsr1 contains a C-terminal CCAAX box, which putatively undergoes both reversible palmitoylation and farnesylation for entry into the secretory pathway. We expressed variants of Rsr1 with mutated C244 or C245, or which lacked GTPase activity (Rsr1K16N and Rsr1G12V), in the rsr1Δ background and compared the resulting phenotypes with those of mutants lacking Bud5 (Rsr1 GEF), Bud2 (Rsr1 GAP), or Cla4. Bud5 was required only for cell size and bud site selection in yeast, suggesting there are alternative activators for Rsr1 in hyphae. Septin ring and vacuole dynamics were restored by expression of unpalmitoylated Rsr1C244S, which localized to endomembranes, but not by cytoplasmic Rsr1C245A or GTP/GDP-locked Rsr1, suggesting Rsr1 functions at intracellular membranes in addition to the plasma membrane. Rsr1K16N or cytoplasmic Rsr1C245A restored normal nuclear division but not septin ring or vacuole dynamics. Rsr1-GDP therefore plays a specific role in suppressing START, which can be signaled from the cytosol. Via differential palmitoylation and activity states, Rsr1 operates at diverse cell sites to orchestrate proper nuclear division and inheritance during constitutive polarized growth. As cla4Δ phenocopied rsr1Δ, it is likely these functions involve Cdc42-Cla4 activity. IMPORTANCE Understanding how single eukaryotic cells self-organize to replicate and migrate is relevant to health and disease. In the fungal pathogen, Candida albicans, the small GTPase, Rsr1, guides the directional growth of hyphae that invade human tissue during life-threatening infections. Rsr1 is a Ras-like GTPase and a homolog of the conserved Rap1 subfamily, which directs migration in mammalian cells. Research into how this single GTPase delivers complex intracellular patterning is challenging established views of GTPase regulation, trafficking, and interaction. Here, we show that Rsr1 directly and indirectly coordinates the spatial and temporal development of key intracellular macrostructures, including septum formation and closure, vacuole dynamics, and nuclear division and segregation, as well as whole-cell morphology by determining branching patterns. Furthermore, we categorize these functions by differential Rsr1 localization and activity state and provide evidence to support the emerging view that the cytosolic pool of Ras-like GTPases is functionally active.

ARP-T1 is a ciliogenesis protein associated with a novel ciliopathy in inherited basal cell cancer, Bazex-Dupré-Christol Syndrome

ABSTRACTActin-Related Protein-Testis1 (ARP-T1)/ ACTRT1 gene mutations cause the Bazex-Dupré-Christol Syndrome (BDCS) characterized by follicular atrophoderma, hypotrichosis and basal cell cancer. Here, we report an ARP-T1 interactome (PXD016557) involved in ciliogenesis, endosomal recycling and septin ring formation. Consequently, ARP-T1 localizes to the midbody during cytokinesis and the basal body of primary cilia in G0. Tissue samples from BDCS patients show reduced ciliary length with significant correlations of ARP-T1 expression levels, confirmed by ACTRT1 knock down. We report that BDCS is a novel ciliopathy and the first case of a skin cancer ciliopathy, where ARP-T1 plays a critical role to prevent pathogenesis.

Transfer of Septin Rings to Cytokinetic Remnants Directs Age-Sensitive ER stress Surveillance Cell Cycle Re-entry

SUMMARYDuring cell division, cells must actively pass on organelles. Previously, we discovered the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway that ensures the inheritance of functional ER. Activation of the ERSU causes the septin ring to mislocalize, which blocks ER inheritance and cytokinesis. Here, we found that the septin ring mislocalizes to previously utilized cell division sites called cytokinetic remnants (CRMs). The transfer of the septin ring to CRMs requires Nba1, a negative polarity component that normally prevents septin ring formation at CRMs. Furthermore, septin ring movement to CRMs relies on the ERSU component Slt2, which is recruited by binding Bem1. During ER stress, Bem1 also binds the GTP exchange factor Cdc24, without activating Cdc42, a GTPase that normally establishes polarized growth. Failure to translocate septin rings to CRMs delays the cell’s ability to re-enter cell division when ER homeostasis is re-established. Thus, ER stress considers the history of previous cell cycle for future cell cycle re-entry upon ER stress recovery.

Mechanisms of MTOC clustering and spindle positioning in the budding yeast Cryptococcus neoformans: a microtubule grow-and-catch model

Mitotic spindle formation in the pathogenic budding yeast, Cryptococcus neoformans, depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before the formation of the mitotic spindle, multiple visible microtubule organizing centers (MTOCs), coalesce into a single focus to serve as an SPB. We propose a ‘grow-and-catch’ model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and catch each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screened various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning - a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintain spatiotemporal spindle localization.Author summaryCells actively self-assemble a bipolar spindle to facilitate chromosomal segregation. Multiple MTOCs, on the outer nuclear envelope, cluster into a single SPB before spindle formation during semi-open mitosis of the budding yeast Cryptococcus neoformans. Eventually, the SPB duplicates and organizes the spindle to position it within the daughter bud near the septin ring during anaphase. In this work, we tested various computational models to match physiological phenomena in an attempt to find plausible mechanisms of MTOC clustering and spindle positioning in C. neoformans. Notably, we propose an MT ‘grow-and-catch’ model that relies on possible redundant mechanisms for timely MTOC clustering mediated by (a) minus end-directed motors that crosslink and slide anti-parallel cMTs from different MTOCs on the nuclear envelope and (b) a Bim1 mediated biased sliding of cMTs along the cell cortex toward the septin ring that pulls MTOCs in the presence of suppressed dynein activity. By combining an analytical model and stochastic MT dynamics simulations, we screened various MT-based forces to detect steady spindle positioning. By screening the outputs of various models, it is revealed that proper spindle positioning near the septin ring requires MT buckling from the cell cortex.

Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1

Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor.” Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.