Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, March 1997. / Also available on the Internet.

No description available.

A general account is given of the development of Daphnia magna from the egg to the mature adult. A review of previous work reveals that authors have concentrated on the early development, neglecting the later development. An adequate technique has been developed. An account of the development based on living material gives a detailed series of stages. A summary of the present state of knowledge of the physiological aspects of development is included. The development of the parthenogenetic egg is described. The early development includes a superficial cleavage and gastrulation by immigration. The mesenteron develops from a solid rod of cells in the ventral part of the egg, and acquires a central cavity which never contains yolk. The yolk cells develop from the blastoderm. The mesoderm develops a single small pair of coelomic cavities, and the heart develops from a compact group of cells. The history of the dorsal organ is described. The development of the ephippial egg resembles that of the parthenogenetic egg except in some features related to the smaller and more even sized nature of the yolk globules. Daphnia magna hatches from the brood pouch of the mother as an immature adult. The account of the anatomy of the adult is a confirmation and extension of previous work, including histology and indicating function. The muscles of the mesenteron are striated, and the heart wall contains anincomplete longitudinal, as well as a circular, layer of muscles. A suggestion is made for a new interpretation of the cells of the branchial sacs. The results obtained and the importance of a large quantity of yolk are discussed.

Cardiac development is a tightly controlled, step-wise process, as the heart is one of the first functional organs to develop. The major cell type of the heart is the cardiomyocyte and is known to be the work horse of the heart. Cardiac myocytes are very different than any other cell type, as they contain an infrastructure that allows for the involuntary contraction of the heart. Beginning at the anchoring of proteins at the z-disk, the sarcomere structure is gradually patterned. By adult, most cardiomyocytes are bi-nucleated, rod-shaped, and terminally differentiated. Although the microtubule network is a key organelle in the differentiation of every cell type, the role of microtubules in the heart, specifically in cardiac myocytes, has not been thoroughly explored. Genetic alteration of the microtubule network by Centromere Protein F (CENP-F) results in several structural and functional abnormalities in both humans and mice. Without CENP-F, mice hearts are dilated and undergo cardiac arrythmias. In isolated cardiomyocytes, calcium influx is disrupted, myocyte stiffness is greatly reduced, and major contractile components are significantly altered. As we observe changes in how CENP-F loss of function differs from cell to cell, our studies underlie the importance of understanding the role of CENP-F in the specific cell types of the organ of interest. Ultimately, these findings are novel as they lay the foundation for further studies on microtubules in cardiac development and health and, provide evidence for the potential impact of microtubule targeted chemotherapy treatments on heart disease.

Proper cell division to yield two daughter cells with identical complements of genomic material requires coordination between mitosis and cytokinesis. In the event of a mitotic error, checkpoint mechanisms must inhibit both mitotic exit and cytokinesis to ensure accurate segregation of chromosomes. In response to mitotic spindle errors, the spindle checkpoint delays cytokinesis by inhibiting the septation initiation network (SIN), a GTPase-driven signaling cascade. Specifically, Hhp1 and Hhp2 phosphorylate the SIN scaffold protein, Sid4, recruiting dimeric the E3 ligase Dma1. Dma1 ubiquitinates Sid4, antagonizing the localization of the Polo-like kinase Plo1, and preventing phosphorylation of its downstream target Byr4. Consequently, the SIN kinase cascade and cytokinesis are delayed. Upon resolution of the mitotic spindle error, the Dma1 checkpoint signal must be withdrawn to allow continuation of the cell division cycle. However, the mechanism by which Dma1 ubiquitination of Sid4 is stopped is not known. Furthermore, whether Dma1 itself is regulated by the checkpoint remains unclear. In this work, I show that Dma1 is post-translationally modified by auto-ubiquitination and phosphorylation. Dma1 exhibits previously unreported localization dynamics that are dependent on its catalytic activity. In addition to ubiquitinating Sid4, Dma1 demonstrates promiscuous Dma1 auto-ubiquitination in vivo, and evidence indicates that its localization dynamics are impaired in the absence of auto-ubiquitination. I also determined that Dma1 is phosphorylated in vivo throughout the cell cycle and that this phosphorylation occurs on seven sites. Furthermore, I demonstrated that Cdk1 and Plo1 can phosphorylate Dma1 in vitro. The Cdk1/Plo1 phospho-mimetic inhibits Dma1 auto-ubiquitination while maintaining checkpoint activity. Overall, the current data point to a model wherein Dma1 auto-ubiquitination decreases its SPB binding and therefore access to its checkpoint substrate. Dma1 phosphorylation at Cdk1 and Plo1 sites prevents auto-ubiquitination, enabling increased SPB localization and maintenance of the checkpoint.

<p> Visual information is relayed from retina to the brain at first order synapses within the lateral geniculate nucleus (dLGN). During development, activity-dependent synaptic competition drives the segregation of retinal ganglion cell terminals into eye-specific zones. It has been assumed that the gain of synaptic transmission within these eye-specific zones is equivalent, providing uniform information transfer from the periphery to the CNS. Here, we revise this understanding. First, we demonstrate that anatomical segregation of retinal axons triggers a profound (200&ndash;300%) potentiation of neurotransmitter release selectively within the projection zone of the ipsilateral eye. Second, optogenetic recruitment of genetically defined axons within the ipsilateral projection zone provides evidence that functional synaptic connectivity is sub-stratified within the ipsilateral dLGN. Thus, we define a new functional organization within the dLGN and propose that synaptic competition acts as a developmental timer that triggers respecification of set point synaptic gain within the ipsilateral dLGN.</p><p>

The intestinal brush border serves as the sole site of nutrient absorption within the body, and also acts as an important barrier against luminal pathogens. The brush border is comprised of membrane protrusions called microvilli that are found on the apical surface of enterocytes. These protrusions are supported by a core bundle of 20 to 30 parallel actin filaments with the plus ends oriented towards the lumen. Our laboratory showed that protocadherin-24 and mucin-like protocadherin play a key role in the assembly and organization of the brush border. These protocadherins interact to form a trans-heterophilic adhesion complex that physically connects the distal tips of microvilli and regulates the tight packing. How these complexes are targeted to microvillar tips remains unknown. Microvillar protocadherins interact with several cytoplasmic binding partners, including the actin based motor, myosin-7b (Myo7b). Given the role of other myosin motors in building actin-based protrusions, this work focuses on elucidating the function of Myo7b in brush border assembly. We show that Myo7b is highly enriched at the tips of microvilli in both kidney and intestinal brush borders. Loss of Myo7b results in the mislocalization of the intermicrovillar adhesion links along the microvillar axis, disrupting brush border assembly. We also found that Myo7b motor domains are capable of supporting tip-directed transport. However, motor activity is supplemented by other passive targeting mechanisms, which together drive highly efficient accumulation of the adhesion complex at the tips. Additionally, we have established an in-cell reconstitution assay using filopodial protrusions that can be used to study protein interactions within the adhesion complex. Using this assay, we have begun to characterize the morphological effects of tip-enriched adhesion on actin-based protrusions. Initial experiments suggest adhesion at the distal tips regulates the stability and length of these protrusions. This work provides evidence that the actin-based motor Myo7b promotes the accumulation of adhesion complexes at microvillar tips, which may alter the morphology and dynamics of actin-based protrusions. These findings hold important implications for understanding apical morphogenesis in transporting and sensory epithelial tissues.

Post-translational modification of proteins are events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). While the mitotic substrates of the APC/C have been well characterized, it is not clear what role deubiquitinating proteases (DUBs) play in antagonizing APC/C function. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast Schizosaccharomyces pombe. Our evidence demonstrates that deletion of the SAGA associated DUB Ubp8 (ubp8Î) suppresses the temperature sensitive phenotypes of APC/C mutants and this suppression is specific to the SAGA DUB module. Furthermore, mutations abolishing histone H2B ubiquitylation were not able to suppress the APC/C temperature sensitive phenotype. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function utilizing a mechanism dependent on H2B ubiquitination. Hhp1/2 are the soluble casein kinase 1 (CK1) family members in S. pombe. One of their functions is to inhibit the septation initiation network (SIN) during a mitotic checkpoint arrest. Hhp1/2 phospho-prime the SIN scaffold protein Sid4 for ubiquitination by the E3 ligase Dma1; Sid4 ubiquitination delays SIN activation and thus cell division. The SIN is assembled at spindle pole bodies (SPBs), and though Hhp1/2 also localize to SPBs, it is not known if their SPB localization is required for SIN inhibition. Here, we establish that Hhp1/2 localize constitutively to SPBs, the nucleus, cell tips, and division site. We find that their catalytic domains but not enzymatic function are used for SPB targeting and that this targeting strategy is conserved in human CK1δ/ε localization to centrosomes. Further, we pinpoint amino acids in the Hhp1 catalytic domain required for SPB interaction; mutation of these residues disrupts Hhp1 association with the core SPB protein Ppc89, and the inhibition of cytokinesis in the presence of spindle stress. Taken together, we have defined a molecular mechanism used by CK1 enzymes to target to a specific cellular locale for compartmentalized signaling.

<p> Celsr1 is an atypical cadherin central to the asymmetric cell-cell complexes that define planar cell polarity (PCP). Previous work has shown that Celsr1 undergoes bulk endocytosis during cell division in the basal layer of the mouse skin. Here, we report the unexpected finding that Celsr1-mediated intercellular complexes remain intact during mitotic internalization, resulting in uptake of Celsr1 and associated PCP components into dividing cells from their neighbors in a process known as trans-endocytosis. Our observations suggest that the bulk of this internalized pool of Celsr1 is targeted for degradation. Furthermore, Celsr1 internalized from neighboring cells carries with it additional core PCP proteins, including the posteriorly-enriched Fzd6 and anteriorly-enriched Vangl2. However surprisingly, Vangl2 originating from the dividing cell is excluded from mitotic endosomes and remains associated with the membrane. Overexpression of Vangl2 in vitro is sufficient to interfere with Celsr1 internalization, and mitotic internalization of Celsr1 within the skin is delayed at anterior cell surfaces. We propose that Vangl2 stabilizes Celsr1 at the membrane and its dissociation from Celsr1 is a prerequisite for Celsr1 turnover. Together our results indicate that mitotic turnover of Celsr1 depends on the displacement of Vangl2 from PCP complexes and results in the non-autonomous turnover of PCP proteins from neighboring cells.</p><p>

<p> Echinoid larvae exhibit phenotypic plasticity, where development of long ciliary bands in low-food conditions is considered advantageous for improved algal particle capture. Conversely, larvae in concentrated algal conditions redirect growth to persisting structures (i.e. not lost during metamorphosis) and develop quickly. This study seeks to understand the organismal growth efficiency of sand dollar larvae (<i>Dendraster excentricus</i>) with different phenotypes by comparing larvae developing in low and high algal concentrations (1,000 and 10,000 algal cells ml^&ndash;1, respectively). I measured ingestion, metabolism, and biomass growth rates (protein and lipid) during larval development in three independent cultures. Resulting phenotypes demonstrated morphological divergence, where high-fed larvae grew smaller arms relative to stomach size (a remaining post-metamorphic feature) compared to low-fed larvae. Physiological data were converted to energetic units (mJ) to determine assimilation and growth efficiencies. Low-fed larvae proportionally allocated more energy to metabolism while the high-fed larvae allocated more toward growth. This resulted in different assimilation and growth efficiencies in low- and high-fed treatments. The energetic demands and organismal growth efficiencies of these contrasting phenotypes is important for understanding the constraints on population connectivity of adult populations in rapidly changing marine conditions.</p><p>