Septins perform diverse functions through the formation of filaments and higher-order structures. However, the exact architecture of septin structures remains unclear. In the budding yeast Saccharomyces cerevisiae, septins form an “hourglass” at the mother-bud neck before cytokinesis, which is converted into a “double ring” during cytokinesis. Here, using platinum-replica electron microscopy, we find that the early hourglass consists of septin double filaments oriented along the mother-bud axis. In the late hourglass, these double filaments are connected by periodic circumferential single filaments on the membrane-proximal side, and are associated with centrally located, circumferential, myosin-II thick filaments on the membrane-distal side. The double ring consists of exclusively circumferential septin filaments. Live-cell imaging studies indicate that the hourglass-to-double ring transition is accompanied by loss of septin subunits from the hourglass, and reorganization of the remaining subunits into the double ring. This work provides an unparalleled view of septin structures within cells and defines their remodeling dynamics during the cell cycle.
SummaryAsymmetric cell division plays a crucial role in cell differentiation, unequal replicative senescence, and stem cell maintenance. In budding yeast, the identities of mother and daughter cells begin to diverge at bud emergence when distinct plasma-membrane domains are formed and separated by a septin ring. However, the mechanisms underlying this transformation remain unknown. Here, we show that septins recruited to the site of polarization by Cdc42-GTP inhibit Cdc42 activity in a negative feedback loop, and this inhibition depends on Cdc42 GTPase-activating proteins. Combining live-cell imaging and computational modeling, we demonstrate that the septin ring is sculpted by polarized exocytosis, which creates a hole in the accumulating septin density and relieves the inhibition of Cdc42. The nascent ring generates a sharp boundary that confines the Cdc42 activity and exocytosis strictly to its enclosure and thus clearly delineates the daughter cell identity. Our findings define a fundamental mechanism underlying eukaryotic cell fate differentiation.
In order to train the neural network for plant phenotyping, a sufficient amount of training data must be prepared, which requires time-consuming manual data annotation process that often becomes the limiting step. Here, we show that an instance segmentation neural network aimed to phenotype the barley seed morphology of various cultivars, can be sufficiently trained purely by a synthetically generated dataset. Our attempt is based on the concept of domain randomization, where a large amount of image is generated by randomly orienting the seed object to a virtual canvas. The trained model showed 96% recall and 95% average Precision against the real-world test dataset. We show that our approach is effective also for various crops including rice, lettuce, oat, and wheat. Constructing and utilizing such synthetic data can be a powerful method to alleviate human labor costs for deploying deep learningbased analysis in the agricultural domain.
Septins, which are involved in cytokinesis, have been identified in a variety of fungi and animal cells. For analysis of the function of septin, drugs targeting septin would be useful; however, no such drugs have been available hitherto. By serendipity, we found that forchlorfenuron (FCF, N-(2-chloro-4-pyridyl)-N-phenylurea, 4PU300), a synthetic plant cytokinin, disturbed cytokinesis in Saccharomyces cerevisiae . Upon administration of FCF, septin structures at the bud neck became deformed and filament-like septin appeared outside of the neck. Under these conditions, the localization of actin was normal and Gin4, which is localized at the bud neck in a septin-dependent manner, was found to remain at the location of apparently normal septin at the bud neck, whereas it was not co-localized to the deformed septin at the bud neck or to septin seen outside the bud neck. FCF administration immediately induced production of sporadic septin structures outside the bud neck, and these structures disappeared promptly upon removal of the drug. Taken together, these findings indicate that FCF maybe a promising drug for investigating the structure and function of septin.
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