Correlation of spindle architecture with dynamic behavior shows that pairs of antiparallel microtubules are sufficient to form a bipolar spindle, whereas interpolar microtubules maintain the speed of pole displacement during spindle assembly. The number of interpolar microtubules formed is controlled in part through γ-tubulin phosphorylation.

Clathrin-mediated endocytosis (CME) is conserved among eukaryotes and has been extensively analyzed at a molecular level. Here, we present an analysis of CME in the human fungal pathogen Candida albicans that shows the same modular structure as those in other fungi and mammalian cells. Intriguingly, C. albicans is perfectly viable in the absence of Arp2/3, an essential component of CME in other systems. In C. albicans, Arp2/3 function remains essential for CME as all 15 proteins tested that participate in CME, including clathrin, lose their characteristic dynamics observed in wild-type (WT) cells. However, since arp2/3 cells are still able to endocytose lipids and fluid-phase markers, but not the Ste2 and Mup1 plasma membrane proteins, there must be an alternate clathrin-independent pathway we term Arp2/3-independent endocytosis (AIE). Characterization of AIE shows that endocytosis in arp2 mutants relies on actin cables and other Arp2/3-independent actin structures, as inhibition of actin functions prevented cargo uptake in arp2/3 mutants. Transmission electron microscopy (TEM) showed that arp2/3 mutants still formed invaginating tubules, cell structures whose proper functions are believed to heavily rely on Arp2/3. Finally, Prk1 and Sjl2, two proteins involved in patch disassembly during CME, were not correctly localized to sites of endocytosis in arp2 mutants, implying a role of Arp2/3 in CME patch disassembly. Overall, C. albicans contains an alternative endocytic pathway (AIE) that relies on actin cable function to permit clathrin-independent endocytosis (CIE) and provides a system to further explore alternate endocytic routes that likely exist in fungal species.

High resolution live cell microscopy is increasingly used to detect cellular dynamics in response to drugs and chemicals, but it depends on complex and expensive liquid handling devices that have limited its wider adoption. Here, we present a microfluidic perfusion system that is built without using specialized microfabrication infrastructure, simple to use because only a pipette is needed for liquid handling, and yet allows for rapid media exchange and simultaneous fluorescence microscopy imaging. Yeast cells may be introduced from a culture, or spotted as arrays on a coverslip, and are sandwiched with a 20 mum thick track-etched membrane. A second coverslip and a mesh with 120 mum porosity are placed on top, forming a microfluidic conduit for lateral flow of solutions by capillary effects. Solutions introduced through the inlet flow through the mesh and chemicals diffuse vertically across the membrane to the cells trapped below. Solutions are exchanged by adding a new sample to the inlet. Using this system, we studied the dynamic response of F-actin in living yeast expressing Sac6-EGFP-a protein associated with discrete F-actin structures called "patches"-to the drug latrunculin A, a well known inhibitor of actin polymerization. We observed that the patches disappeared in 85% of the cells within 5 min, and re-assembled in 45 min following exchange of the drug with media. The perfusion system presented here is a simple, inexpensive device suited for analysis of drug dose-response and regeneration of single cells and arrays of cells.

Highlights d The monopolar-to-bipolar spindle transition is fast and irreversible d The fast transition is driven by Cin8 (Kinesin-5) microtubule crosslinking d Nascent bipolar spindles need Kinesin-5 sliding for steadystate lengths >1 mm d Spindle formation sequentially integrates Kinesin-5 MT crosslinking and sliding

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