ORF45 of Kaposi's sarcoma–associated herpesvirus (KSHV) is a gamma herpesvirus-specific, immediate-early, and tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other co-purified proteins, including prominent ones of ~38 kDa and ~130 kDa. Mass spectrometry revealed that the 38 kDa protein is viral ORF33 and the 130 kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl terminal 19-aa of ORF45, and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7, in both transfected conditions and KSHV-infected cells. Moreover, we noticed an ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation, which was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. To determine if the binding of ORF33 is a critical function of C19, we used co-precipitation with point mutants of the C19 region and identified two required residues: tryptophan 403 and tryptophan 405. We then engineered KSHV genomes containing these mutants and transfected them into iSLK cells. Similar to the C19 deletion mutant, we found that both binding-deficient mutants exhibited decreased ORF33 accumulation and viral particle production. Since C19 is sufficient for binding ORF33, we hypothesized that introduction of a C19 analogue could inhibit binding and may lead to a similar decrease in viral particle production. We used ELISA to measure the binding of ORF33 to ORF45 in the presence of TAT-C19 and found that TAT-C19 inhibited binding in a dose-dependent manner. To measure the analogue's effect on viral particle production, we treated KSHV-infected cells with TAT-C19 and found that TAT-C19 inhibited viral particle production in a dose-dependent manner. In conclusion, binding of ORF33 and ORF45 during the lytic cycle is required for accumulation of the ORF33 and production of viral particles. In addition to forming a complex with ORF33 and USP7, we also found a strong association of ORF45 to RSK and ERK during the lytic cycle, matching previous reports that ORF45 bound ERK and RSK in a single complex. During that study, ORF45 was found to form a complex with ERK and RSK and the formation of this complex lead to accumulation of active ERK and RSK. While the binding site of RSK was mapped to aa 55-70, it was unclear if ERK bound to ORF45 directly or through RSK. Using in vitro binding analysis, we identified an ERK binding site in aa 16-35. Using T-Coffee analysis, we compared the sequences of gamma-2 herpesvirus homologues of ORF45 and found two highly conserved phenylalanine residues at aa 32 and 34. After generating point mutants of each residue to alanine, we measured their effect on the activation of ERK and RSK induced by ORF45 and found that either mutation lead to decreased activation of ERK and RSK. We are currently evaluating their effect upon the activation of ERK and RSK during the lytic cycle and the production of progeny virions. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the Doctor of Philosophy. / Fall Semester 2015. / November 3, 2015. / ERK1/2, Kaposi's sarcoma-associated herpesvirus, KSHV, ORF33, ORF45, p90RSK / Includes bibliographical references. / Fanxiu Zhu, Professor Directing Dissertation; Scott Stagg, University Representative; Hengli Tang, Committee Member; Thomas C. S. Keller, III, Committee Member; Kathyrn M. Jones, Committee Member.

Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus that has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on KSHV progeny virion production. Importantly, we investigated the regulation of gene expression by ORF45-actvated RSK by performing RNA-seq of KSHV-infected cells. We show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. One of the few viral substrates of ORF45-activated identified by our mass spectrometry analysis was ORF36. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. We report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in their binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N-terminus of ORF36 or an acidic patch of ORF45 abolished the binding. Additionally, dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we employed BAC mutagenesis to engineer both ORF36-null and kinase-dead (KD) mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45, and indicate a significant role of ORF36 in the production of infectious progeny virions. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates viral and cellular kinase signaling to optimize lytic replication. The findings reported here have important implications for the pathobiology of KSHV and other diseases in which RSK activity is missregulated. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2016. / March 22, 2016. / Kaposi's sarcoma, kinase, KSHV, lytic replication, ORF45, RSK / Includes bibliographical references. / Fanxiu Zhu, Professor Directing Dissertation; Qing-Xiang Sang, University Representative; Hengli Tang, Committee Member; David Gilbert, Committee Member; Jonathan H. Dennis, Committee Member.

Microfluidic devices are widely used for cell-based analysis. There are always needs to develop simpler, more effective and/or less costly devices than the existing ones for this application. A simple microfluidic device has been fabricated and tested for studying chemotaxis of macrophages in this study. The device was made of polydimethylsiloxane bound to a cell culture dish. It consisted of a millimeter-sized cavum and two arrays of straight channels of 5 um in width and 6um height and about two millimeters in length. The channels connected the cavum, in which a chemoattractant was loaded, with the surrounding environment, in which the macrophages were cultured. The device was first tested with a known chemoattractant - fetal bovine serum and the chemoattractive property of myelin basic protein (MBP) was then studied using the device. The macrophages were found to migrate towards to the MBP-loaded cavum in larger quantity and greater distance than those in the control samples. The results prove the usefulness of the microfluidic device for chemotaxis assay and indicate that MBP is a chemoattractant for the macrophages. / A Thesis submitted to the Department of Chemical and Biomedical Engineering in partial fulfillment of the 2017. / Summer Semester 2017. / July 20, 2017. / macrophage, Microfluidic systems, myelin basic protein, PDMS / Includes bibliographical references. / Hoyong Chung, Committee Member; Hadi Mohammadigoushki, Committee Member.

Cells sense and respond to topographical cues in their microenvironment that influence growth, development, and migration. Cell migration and outgrowth assays have been used to study cellular movement or changes in cellular morphology and topography. Such assays are promising tools in drug discovery, especially when implemented with high-throughput and high-content imaging systems. These techniques have also been useful for screening and analyzing the effect of different compounds on neurite outgrowth and topography which in turn may lead to the discovery of beneficial targets for regeneration of nervous tissue. Typically, high-throughput screening of large chemical libraries is employed during the early stages of discovering new drug entities. However, these screening assays do not utilize different topographical surfaces. Many common techniques such as the scratch wound assay are limited in their compatibility with patterned surfaces. Therefore, there is a need to develop novel technologies capable of identifying potentially therapeutic compounds in early stage of drug discovery processes that can regulate cell behaviors and are not limited in their throughput and compatibility with patterned surfaces. A potentially scalable approach is the “fence” assay in which cells are cultured on topographical surfaces which are partially covered by a removable barrier. Upon removal of the barrier, cells are free to spread and migrate on the freshly uncovered topographies. In this thesis, a novel technique called evaporative edge lithography (EEL) is demonstrated as an approach to miniaturize the fence assay and can be used for high-throughput screening (HTS) in early stages of drug discovery. Furthermore, EEL is a new method to fabricate lipid-based drug delivery microarrays. Lipid multilayer micro-patterns offer a promising approach to applications such as drug screening and biosensing that require well defined patterns and fluidity. It is shown in this thesis that the factors that govern stability and instability of lipid multilayer nanostructures upon immersion using fluorescence microscopy and observed the following four mechanisms of lipid multilayer instability and strategies are derived to control immersion stability based on these findings: (1) Dissolution by the air/water interface; (2) Disruption by shearing from flowing solution; (3) Spreading at the solid-liquid interface; (4) Diffusion into solution. Based on these studies, a lipid multilayer microarray was developed that is suitable for cell-based assays without detectible cross-contamination by culturing cells on lipid patterns. It is shown in this thesis that this assay is compatible with poorly soluble lipophilic drug compounds that pose a challenge for HTS microarray assays. EEL was demonstrated for topographically patterned surfaces for screening compounds on adherent cells. Lipophilic compounds including docetaxel and BFA were screened using EEL with cultured HeLa cells to test if migration is affected and can be quantified with this approach. These results indicate that docetaxel and BFA were delivered locally into cells locally from surface supported lipid films and significantly inhibited cellular migration. Subsequently, EEL was used to screen docetaxel on cultured primary olfactory bulb neuronal cells to test the effect on neurite outgrowth. EEL is a novel approach that allows delivery and subsequent study of the effects of poorly water-soluble drugs on cell migration as well as in vitro screening of different drugs for their effects on cell structures and functions. In addition, this migration assay is a scalable and promising approach for high throughput drug screening microarrays since multiple drug compounds at different dosages can be screened simultaneously on the same surface. This work will advance future studies in developing a portable assay capable of screening lipophilic cancer and neurotropic compounds for topographically-driven cell outgrowth and migration. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / April 5, 2018. / cell migration, drug screening, evaporative edge lithography, lipid multilayers, lipid patterning, neurite outgrowth / Includes bibliographical references. / Steven Lenhert, Professor Directing Dissertation; Jingjiao Guan, University Representative; Kathryn Jones, Committee Member; Thomas Keller, Committee Member; Jonathan Dennis, Committee Member.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of significant public health concern closely related to other highly pathogenic flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). With the rise of ZIKV in Brazil in 2015, its potential link to microcephaly and other severe neurological birth defects prompted the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. Since this time, numerous studies have provided ample evidence to establish ZIKV as the causative agent of microcephaly, yet the molecular mechanisms underlying these neurodevelopmental defects are not well understood. We therefore establish a tractable experimental model system to investigate the impact of ZIKV on human neural development. We demonstrate that ZIKV efficiently infects human cortical neural progenitor cells (hNPCs) derived from induced pluripotent stem cells, but less efficiently infects other cells along the neural differentiation pathway, including immature cortical neurons. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection disrupts cell cycle progression and induces cell death in hNPCs contributing to their attenuated growth. Global transcriptome analyses of ZIKV-infected hNPCs reveal transcriptional dysregulation, notably a downregulation of cell-cycle-related genes, highlighting the potential involvement of cell cycle pathways in ZIKV biology. We then study the molecular mechanisms by which ZIKV manipulates the cell cycle in hNPCs and the functional consequences of cell-cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that host cell-cycle disruption is unique to ZIKV among the flaviviruses tested, including DENV and WNV, however similar among the two strains of ZIKV tested, including the prototype Uganda strain and a Puerto Rican strain. ZIKV, but not DENV, infection induces DNA double-strand breaks, triggering the DNA damage response through the ATM/Chk2 signaling pathway, while suppressing activation of the ATR/Chk1 signaling pathway in hNPCs. Furthermore, ZIKV infection impedes the progression of cells through S phase thereby preventing the completion of host DNA replication. Recapitulating the S-phase arrest state with S-phase inhibitors leads to an increase in ZIKV replication, but not of WNV or DENV replication. Together, our results identify hNPCs as a direct target of ZIKV and the damaging impact of ZIKV on the growth of hNPCs. Importantly, our data demonstrate ZIKV’s ability to induce host DNA damage and arrest cell cycle progression, which results in a cellular environment favorable for its replication. As hNPCs generate the cortical neurons during early fetal brain development, the ZIKV-mediated growth retardation likely contributes to the neurodevelopmental defects of the congenital Zika syndrome. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / January 23, 2018. / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Timothy Megraw, University Representative; Brian P. Chadwick, Committee Member; David M. Gilbert, Committee Member; Yan Li, Committee Member; Fanxiu Zhu, Committee Member.

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding. Misfolded proteins are aggregation prone and protein aggregation are associated with many human diseases, such as neurodegenerative disorders and cancer. However, the molecular basis underlying the proteotoxicity and the mechanisms to combat this toxicity remain poorly understood. We used S. cerevisiae, budding yeast, as a model organism to address these questions as much of the protein homeostasis machinery is conserved form yeast to humans. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which is responsible for the development of Huntington's disease, a neurodegenerative disorder. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and degradation of misfolded proteins. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Our previous data indicate that the degradation of mutated Htt with a 103 polyQ expansion (Htt103QP) depends on both the ubiquitin proteasome system and autophagy in budding yeast. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells. We showed that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. In addition, mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Notably, we detected more aggregated forms of Htt103QP in sse1∆ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance but are less critical for proteasome-dependent degradation. These findings uncover the role of HSPs in the inclusion body formation and autophagy-mediated clearance of mutated Huntingtin. Using budding yeast as a model system, we further asked why misfolded proteins are toxic, and how eukaryotic cells combat this toxicity. Our results support the notion that ubiquitinated misfolded protein aggregates drain free ubiquitin and compromise ubiquitin-dependent protein degradation, but the AAA+ ATPase Cdc48 counteracts the toxicity by segregating these protein aggregates. Using Htt103QP as model misfolded protein, we found that Cdc48 and its two predominant cofactors, Npl4 and Ufd1, are required for the segregation and degradation of Htt103QP in yeast cells. We also identified the E3 ubiquitin ligase San1 that catalyzes Htt103QP ubiquitination and facilitates its proteasome-dependent degradation. Unexpectedly, deletion of San1 and another ubiquitin ligase, Ubr1, suppressed the growth defects and accumulation of ubiquitinated substrates in cells lacking functional Cdc48Ufd1/Npl4. We further show compromised ubiquitin-proteasome system in cdc48 mutants, as well as the suppression of these defects by san1∆ ubr1∆, indicating that ubiquitination of misfolded proteins contributes to the growth defect in cdc48 mutants. Importantly, we found that overexpression of ubiquitin partially rescued the growth defects in cdc48 mutants. Finally, we showed that blocking ubiquitination of misfolded proteins by san1∆ ubr1∆ increases the resistance of yeast cells to some proteotoxic stressors. Our results reveal the basis for the cytotoxicity of misfolded proteins and highlight the role of Cdc48 in alleviating this toxicity. Lastly, we showed the ubiquitin ligase Rsp5 is necessary for the inclusion body formation and autohagic degradation of Htt103QP. Also, cells with defective Rsp5 exhibit compromised K63-linked ubiquitination of Htt103QP indicating K63-linked ubiquitination could facilitate autophagic clearance of Htt103QP. Supporting this notion, cells expressing a mutant form of ubiquitin (K63R) that are unable to promote K63-linked ubiquitination exhibit Htt103QP IB defect. Rsp5 has also been implicated in ubiquitinating the autophagy adapter protein Cue5. We showed yeast cells lacking Cue5 exhibit Htt103QP autophagy defect which is consistent with published data that used a different mutated Huntingtin protein. Taken together, our research work identified several components in the cellular response to misfolded proteins. We identified a several factors required for mutated Huntingtin inclusion body formation and autophagic degradation. Also, we uncovered a mechanism that can explain why misfolded proteins are cytotoxic and how cells combat this toxicity. These findings may provide novel targets in developing strategies to combat protein misfolding diseases and cancer. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2018. / November 16, 2018. / Includes bibliographical references. / Yanchang Wang, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Timothy Megraw, Committee Member; Yi Zhou, Committee Member; Akash Gunjan, Committee Member.

Karenia brevis is a marine dinoflagellate commonly found in the Gulf of Mexico and important both ecologically and economically due to its production of the neurotoxin brevetoxin, which can cause respiratory illness in humans and widespread death of marine animals. K. brevis strains have previously shown to be sensitive to changes in CO2, both in terms of growth as well as toxin production. Our study aimed to understand this sensitivity by measuring underlying mechanisms, such as photosynthesis, carbon acquisition, and photophysiology. K. brevis (CCFWC-126) did not show a significant response in growth, cellular composition of carbon and nitrogen, nor in photosynthetic rates between pCO2 concentrations of 150, 400 or 780 µatm. However, a strong response in its acquisition of inorganic carbon was found. Half saturation values for CO2 increased from 1.5 to 3.3 µM, inorganic carbon preference switched from HCO3- to CO2 (14% to 56% CO2 usage), and external carbonic anhydrase activity was downregulated by 23% when comparing low and high pCO2. We conclude that K. brevis must employ an efficient and regulated carbon concentration mechanism (CCM) to maintain constant carbon fixation and growth across pCO2 levels. A positive correlation with pCO2, although not statistically significant, in cellular brevetoxin content was found. This study is the first explaining how this socioeconomically important species is able to efficiently supply inorganic carbon for photosynthesis which can potentially prolong bloom situations. This study also highlights that enhanced CO2, as projected for a future ocean, can affect underlying physiological processes of K. brevis, some of which could lead to increases in cellular brevetoxin production and therefore increased impacts on coastal ecosystems and economies. / A Thesis submitted to the Department of Earth, Ocean, and Atmospheric Science in partial fulfillment of the requirements for the degree of Master of Science. / Fall Semester 2018. / September 17, 2018. / Brevetoxins, Carbon Concentrating Mechanism, Climate Change, Ocean Acidification, Photosynthesis, Red Tides / Includes bibliographical references. / Sven Kranz, Professor Directing Thesis; Angela Knapp, Committee Member; Olivia Mason, Committee Member; Michael Stukel, Committee Member; Janie Wulff, Committee Member.

Endocytosis, or cell 'eating,' is a process used by cells for functions such as ingesting foreign particles during an immune response, sensing environmental cues during development and fine-tuning communication between synapses during a neuronal transmission. Endocytosis also allows individual cells to internalize their own protein and membrane components from the plasma membrane to maintain polarized growth, recycle membrane-bound receptors and perform quality control within the cell by guiding damaged proteins for degradation. The unicellular model organism, fission yeast, is an excellent system to study conserved aspects of endocytosis. Clathrin-mediated endocytosis, the focus of this thesis, involves the formation of a clathrin coat and polymerization of a branched actin network around the endocytic site, which facilitates internalization of the plasma membrane. The assembly of over 50 proteins during this process occurs under a minute with very high precision. However not much is known about the precise temporal regulation of these steps. In this thesis I report the discovery of a switch that regulates the timing of actin polymerization during endocytosis. I characterize a novel component of the endocytic machinery, dip1p, which is involved in regulating this switch. I highlight additional modes of activation of actin polymerization and endocytosis in dip1 mutants. In my assessment for the requirement for actin polymerization during endocytosis, I discover that one role of actin polymerization during the initial invagination step of endocytosis in yeast is to overcome the tremendous turgor pressure within the cell. I show that in certain mutants defective in actin polymerization, defects in endocytosis can be rescued by reducing turgor pressure. I also show that the cell wall does not contribute to forces required for endocytic internalization. Finally, I report the fortuitous sighting of filamentous actin in the nuclei of a certain mutant fission yeast cell, namely dip1for3 double mutant cells. An excess of nuclear actin leads to defects in nuclear architecture and appears to cause defects in chromosome segregation. This finding establishes a model system to gain further insight into functions of nuclear actin. In summary, this thesis provides insights into the requirement and novel mechanisms of regulation of Arp2/3-mediated actin polymerization and furthers the understanding of mechanisms of endocytosis. These discoveries can form the basis for further studies in other conserved processes, such as cell migration, microbial pathogenesis and cell division that require polymerization of actin filaments.

In the following chapters, I describe factors underlying actin cable dynamics and assembly in the budding yeast, S. cerevisiae. First, I examined the role of type II myosin and a tropomyosin isoform in retrograde actin flow (Chapter II). In yeast and other cell types, actin undergoes retrograde or centripetal movement from the cell cortex towards the interior of the cell. Retrograde actin flow drives intracellular and cellular movement. Previous work in the Pon laboratory showed that actin cables undergo retrograde flow, which occurs, in part, from the force generated from actin polymerization and assembly at the elongating filament end. First, we find that the type II myosin, Myo1p, facilitates retrograde flow. We found that the rate of retrograde actin cable flow is reduced by 1) deletion of Myo1p, 2) displacement of Myo1p from the bud neck, or 3) a conditional mutation that inhibits Myo1p motor activity. These findings indicate that myosin motor activity provides the pulling force to drive movement of elongating actin cables from their site of assembly in the bud tip toward the mother cell. Additional work found that a tropomyosin isoform, Tpm2p, negatively regulates retrograde flow through inhibition of type II myosin binding to F-actin within actin cables. Since type II myosins and tropomyosins have a similar function in retrograde actin flow in animals cells, these findings provide the first evidence that yeast can be used as a model system to study this fundamental, conserved mechanism for actin dynamics. Second, I conducted a drug-based screen for novel regulators of actin cables (Chapter III). Previous studies revealed a role for the yeast formins (Bni1p and Bnr1p) in stimulating polymerization of F-actin for actin cable formation, elongation and retrograde flow, and for other actin cable constituents including tropomyosins and actin bundling proteins in stabilizing and organizing F-actin within actin cables. Earlier work has revealed both that actin cables are selectively destabilized by low levels of the actin-destabilizing drug Latrunculin-A (Lat-A), and this drug inhibits cell growth. I carried out a screen designed to identify non-essential gene deletions that reduce the sensitivity of yeast to the growth inhibiting effects of low doses of Lat-A. Eighteen out of 4,848 deletion strains comprising the yeast deletion library exhibited reduced sensitivity to low levels of Lat-A. Eight of the genes represent uncharacterized open reading frames (ORFs) or encode proteins with no known function or activity. Deletion of a majority of these gene results in increased actin cable number. Additionally, I found the growth inhibiting effects of Lat-A are not suppressed by 1) overexpression of either of TPM1 or TPM2 or 2) deletion of TPM2 and the associated increase in the rate of retrograde actin cable flow. Moreover, I found that one of the genes that reduces the growth-inhibiting effects of Lat-A, YHR022c, is an uncharacterized ORF which encodes a novel Ras-like protein. We call this gene Rar1p for Ras-like actin cable regulator. I found that deletion of RAR1 or expression of a constitutively active formin (Bni1p) produces similar phenotypes: 1) increased actin cable content in the presence and absence of low levels of Lat-A, 2) increased retrograde actin cable flow rates, and 3) resistance to Lat-A-dependent inhibition of growth. Finally, I found that the increase in actin cable content observed upon deletion of RAR1 requires Bni1p and not Bnr1p. Our findings reveal a role for previously uncharacterized genes in the regulation of actin cable stability, and new roles for previously characterized, conserved genes in this process. Equally important, I identified a novel Ras-like protein, Rar1p, and found that it affects actin cable abundance and sensitivity to Lat-A by functioning as an isoform-specific, negative regulator of the formin protein Bni1p. Chapter IV describes future directions for the work outlined in chapters II and III.

Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the budding yeast, Saccharomyces cerevisiae. Recent studies have revealed a checkpoint that inhibits cytokinesis when there are severe defects in mitochondrial inheritance. An established checkpoint signaling pathway, the mitotic exit network (MEN), participates in this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelle inheritance checkpoints for mitochondria and other organelles.