Mechanisms of nuclear segregation in the multinucleate and multibudding yeast, Aureobasidium pullulans

Abstract

Aureobasidium pullulans is a ubiquitous polyextremotolerant fungus exhibiting diverse morphologies and growth modes including typical single budding yeast and unconventional large multinucleate yeast able to form multiple buds within a single cell cycle. This complexity presents an exciting opportunity to explore novel aspects of cell biology, especially in the context of nuclear dynamics and cytoskeletal organization. However, genetic and imaging tools to study this organism's cell biology have been limited. Here, we describe the development of foundational cell biology tools for A. pullulans, enabling the expression and visualization of fluorescent probes for nuclei and cytoskeletal components in live cells. Using these tools, we provide the first detailed characterization of mitosis in a multinucleate, multibudding yeast. We observe highly synchronous mitoses that occur in a “semi-open” manner in which the nuclear envelope becomes permeable but remains intact. Strikingly, unlike in the well-studied budding yeast, Saccharomyces cerevisiae, Candida albicans, Ustilago maydis, or Cryptococcus neoformans, mitotic spindles in A. pullulans do not pre-align along the mother-bud axis, and nuclear segregation often occurs after spindle disassembly. Cortical Num1-dynein mediated forces pull highly mobile nuclei into the buds, which hinder the entry of a second nucleus, leading to most buds containing a single nucleus. These findings reveal significant departures from typical model yeast mitosis and suggest that A. pullulans is a powerful new model for investigating cell division and nuclear movement in complex cellular geometries.