Delineating the Roles of Kinesin and Dynein Motor Proteins During Mitotic Spindle Assembly and Function in the Fungal Pathogen Candida albicans

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Shoukat, Irsa
Keyword
Fungal pathogens , Kinesin , Dynein , Motor proteins , Candida albicans , Budding yeast , Cell Biology , Microbiology , Antifungal Drug Resistance
Abstract
During cell division, cells must accurately orchestrate and execute DNA segregation to daughter cells. To do this, eukaryotes assemble a dynamic macromolecular structure called the mitotic spindle, which undergoes a series of conformational changes to attach, orient, and separate sister chromatids. These dynamic events depend on the correct localization and activation of structurally and functionally diverse teams of microtubule-associated motor proteins. Kinesins and dyneins are the main families of motors involved in orienting and moving the microtubule arrays that compose, or are attached to, the mitotic spindle. Many of these motor proteins' roles and molecular mechanisms have been extensively studied in several model systems. These studies have led to the broad perception that some motor functions have been evolutionarily conserved. One example is that bipolar spindle assembly ubiquitously requires kinesin-5 family members and thus these motors are essential for cell viability. However, we discovered that the human fungal pathogen, Candida albicans, relies heavily on the kinesin-14 motor, Kar3Cik1, for bipolar spindle assembly, rather than its kinesin-5, Kip1. This suggests that C. albicans uses force-producing motors differently than other eukaryotes and even related yeasts. The studies detailed in this thesis's first two experimental chapters have delineated the roles of Kip1 and dynein (Dyn1) in C. albicans mitosis. Chapter 3 demonstrates that cells lacking Kip1 can undergo all mitotic processes, albeit with subpar integrity. Kip1-deficient cells have shorter metaphase spindles and longer and more numerous astral microtubules. Interestingly, Kip1 mutants are delayed in their entry into anaphase but execute anaphase at the same rate as wild-type cells. Furthermore, simultaneous loss of function of Kip1 and Kar3Cik1 is lethal. Chapter 4 provides evidence that Kip1 works collaboratively with Dyn1 in spindle elongation and that either motor can partially compensate for the loss of the other. The final experimental chapter, Chapter 5, investigates the curious phenotype that some spindles in Kip1 mutants disintegrate and form duplicated bipolar spindles within the same cell compartment. Evidence is provided that subsequent division of these spindles leads to the generation of random aneuploidies that can provide a growth advantage compared to wild-type cells when exposed to the antifungal drug fluconazole. Transcriptomic analysis of fluconazole-treated cells showed altered expression patterns of several genes involved in mitosis, including KIP1. This finding suggests that C. albicans cells may actively regulate mitotic kinesins under stress to promote spindle errors that foster aneuploidy and act as a driver of adaptive genome plasticity.
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