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Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

by Chow Fung-cheung, Judy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 106-117). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF ABBREVIATIONS --- p.ix / LIST OF FIGURES AND TABLES --- p.xii / Chapter SECTION I- --- ISOLATION OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1 --- Functions of Actin --- p.1 / Chapter 1.1.1 --- Functions of Actin in Animals --- p.1 / Chapter 1.1.2 --- Functions of Actin in Plants --- p.2 / Chapter 1.1.2.1 --- In Lower Plants --- p.2 / Chapter 1.1.2.2 --- In Higher Plants --- p.2 / Chapter 1.2 --- Molecular Studies of Actin Gene Families in Plants --- p.4 / Chapter 1.2.1 --- Multigene Family --- p.4 / Chapter 1.2.2 --- Homologies Across Kingdom --- p.4 / Chapter 1.2.3 --- Homologies Within Kingdom --- p.5 / Chapter 1.2.4 --- Position of Intron --- p.5 / Chapter 1.2.5 --- Differential Expression of Actin Genes --- p.7 / Chapter 1.3 --- Objectives of Present Studies --- p.7 / Chapter CHAPTER 2: --- GENERAL TECHNIQUES / Chapter 2.1 --- Growth of Algal Strain --- p.9 / Chapter 2.2 --- Growth of Bacterial Strains --- p.10 / Chapter 2.3 --- Agarose Gel Electrophoresis --- p.10 / Chapter 2.4 --- Restriction Enzyme Digestion --- p.10 / Chapter 2.5 --- Recovery of DNA Fragments from Agarose Gel --- p.11 / Chapter 2.5.1 --- Glass Powder Elution of DNA --- p.11 / Chapter 2.5.2 --- Sephaglas´ёØ BandPrep Kit --- p.11 / Chapter 2.6 --- Large Scale Preparation of Plasmid by Using Magic´ёØ Maxipreps DNA Purification System --- p.12 / Chapter 2.7 --- Ligation --- p.13 / Chapter 2.8 --- Preparation of Competent Cells --- p.13 / Chapter 2.9 --- Transformation of Competent Cells --- p.14 / Chapter 2.10 --- Screening of Recombinant Plasmids --- p.14 / Chapter 2.11 --- Spun Column Techniques --- p.15 / Chapter CHAPTER 3: --- PCR-CLONING OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter 3.1 --- Introduction --- p.16 / Chapter 3.2 --- Materials and Methods --- p.16 / Chapter 3.2.1 --- Preparation of Genomic DNA from C. vulgaris --- p.16 / Chapter 3.2.2 --- Amplification of Actin Genomic Fragments by PCR --- p.17 / Chapter 3.2.3 --- Cloning of PCR Products --- p.17 / Chapter 3.2.4 --- Southern Blotting --- p.18 / Chapter 3.2.5 --- Radiolabeling of DNA Probe --- p.19 / Chapter 3.2.6 --- Prehybridization and Hybridization --- p.19 / Chapter 3.2.7 --- Sequencing Strategies --- p.20 / Chapter 3.2.7.1 --- Isolation of Template DNA --- p.20 / Chapter 3.2.7.2 --- Template Denaturation and Primer Annealing --- p.21 / Chapter 3.2.7.3 --- Labeling and Termination Reaction --- p.21 / Chapter 3.2.7.4 --- DNA Sequencing Electrophoresis --- p.21 / Chapter 3.3 --- Results and Discussion --- p.22 / Chapter CHAPTER 4: --- CLONING OF ACTIN COMPLEMENTARY DNA FRAGMENT FROM CHLORELLA VULGARIS / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Materials and Methods --- p.31 / Chapter 4.2.1 --- Preparation of RNA --- p.31 / Chapter 4.2.2 --- RT-PCR --- p.32 / Chapter 4.2.3 --- Southern Blotting and Hybridization --- p.32 / Chapter 4.2.4 --- Radiolabeling of DNA Probe --- p.32 / Chapter 4.2.5 --- Cloning of RT-PCR Product --- p.33 / Chapter 4.2.6 --- DNA Sequencing --- p.33 / Chapter 4.2.7 --- Sequence Analysis --- p.33 / Chapter 4.3 --- Results and Discussion --- p.34 / Chapter CHAPTER 5: --- SEQUENCE COMPARISON OF ACTIN GENES / Chapter 5.1 --- Introduction --- p.44 / Chapter 5.2 --- Materials and Methods --- p.44 / Chapter 5.3 --- Results and Discussion --- p.44 / Chapter 5.3.1 --- Nucleotide Sequence Analysis --- p.44 / Chapter 5.3.2 --- Analysis of the Predicted Amino Acid Sequence --- p.49 / Chapter 5.3.3 --- Codon Usage --- p.49 / Chapter 5.3.4 --- Intron-Exon Structure in Plant Actin Genes --- p.51 / Chapter 5.3.5 --- General Discussion --- p.53 / Chapter CHAPTER 6: --- ISOLATION OF FURTHER UPSTREAM SEQUENCE FOR ACTIN GENE (CAc18G) FROM CHLORELLA VULGARIS / Chapter 6.1 --- Introduction --- p.54 / Chapter 6.2 --- Materials and Methods --- p.54 / Chapter 6.2.1 --- Genomic Southern Analysis --- p.54 / Chapter 6.2.2 --- Preparation of Actin-Enriched DNA Fraction --- p.55 / Chapter 6.2.3 --- Ligation of Actin-Enriched Fragments with Specific DNA Cassette --- p.55 / Chapter 6.2.4 --- Amplification of Upstream Sequence by Nested PCR --- p.55 / Chapter 6.2.5 --- DNA Sequencing --- p.56 / Chapter 6.3 --- Results and Discussion --- p.57 / Chapter SECTION II - --- CONSTRUCTION OF TRANSGENIC CASSETTES FOR THE PRODUCTION OF BACILLUS TOXIN IN CHLORELLA VULGARIS / Chapter CHAPTER 1： --- INTRODUCTION / Chapter 1.1 --- Characteristics of Algae --- p.64 / Chapter 1.2 --- Biotechnology Potential of Algae --- p.66 / Chapter 1.3 --- Transgenic Algae --- p.68 / Chapter 1.3.1 --- Genes of Selection for Transformant --- p.70 / Chapter 1.3.1.1 --- Homologous Genes --- p.70 / Chapter 1.3.1.2 --- Heterologous Genes --- p.70 / Chapter 1.3.2 --- Transformation Technologies Used in Algae --- p.71 / Chapter 1.3.3 --- Expression of Transgenes in Algae --- p.73 / Chapter 1.4 --- Bacillus Toxin --- p.73 / Chapter 1.4.1 --- Bacillus thuringiensis --- p.73 / Chapter 1.4.2 --- Classification of Bacillus Toxin Genes (Cry Genes) --- p.74 / Chapter 1.4.2.1 --- Type I (CtyI Genes) --- p.74 / Chapter 1.4.2.2 --- Type II (CryII Genes) --- p.75 / Chapter 1.4.2.3 --- Type III (CryIII Genes) --- p.76 / Chapter 1.4.2.4 --- Type IV (CryIV Genes) --- p.76 / Chapter 1.4.3 --- Mode of Action of Insecticidal Effects --- p.77 / Chapter 1.5 --- Insect-Resistance Transgenic Plants --- p.78 / Chapter 1.5.1 --- Transgenic Plants Expressing Crystal Protein Gene --- p.79 / Chapter 1.5.2 --- Problems Encountered --- p.80 / Chapter 1.6 --- Aims of Present Studies --- p.80 / Chapter CHAPTER 2: --- CONSTRUCTION OF TRANSGENIC CASSETTES / Chapter 2.1 --- Introduction --- p.82 / Chapter 2.2 --- Materials and Methods --- p.82 / Chapter 2.2.1 --- Preparation of Plasmids Involved in the Construction of Master Cassette --- p.82 / Chapter 2.2.2 --- Construction of Master Cassette --- p.83 / Chapter 2.2.3 --- Multiple Cloning Site (MCS) of Master Cassette --- p.83 / Chapter 2.2.4 --- Preparation of Plating Cells --- p.85 / Chapter 2.2.5 --- Titering --- p.85 / Chapter 2.2.6 --- Preparation of Plate Lysate --- p.86 / Chapter 2.2.7 --- Amplification of Coding Region of CryIVC Gene --- p.86 / Chapter 2.2.8 --- Cloning of PCR Products --- p.87 / Chapter 2.2.9 --- Construction of Transgenic Cassette --- p.87 / Chapter 2.2.10 --- Confirmation of the Junction Sites --- p.89 / Chapter 2.2.11 --- Testing for the Sensitivity of Algae Towards Kanamycin --- p.90 / Chapter 2.3 --- Results and Discussion --- p.91 / Chapter CHAPTER 3: --- TRANSFORMATION OF ALGAE BY ELECTROPORATION / Chapter 3.1 --- Introduction --- p.97 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Harvesting of Algae --- p.97 / Chapter 3.2.2 --- Electroporation at Different Field Strength --- p.98 / Chapter 3.2.3 --- Plating Culture of Algal Cells --- p.98 / Chapter 3.2.4 --- Preparation of Plasmids for Electrop oration --- p.98 / Chapter 3.2.5 --- Transformation of Algae --- p.100 / Chapter 3.2.6 --- Study on the Uptake of DNA after Electrop oration --- p.100 / Chapter 3.2.6.1 --- Genomic DNA Preparation --- p.100 / Chapter 3.2.6.2 --- Analysis of DNA Uptake --- p.101 / Chapter 3.3 --- Results and Discussion --- p.101 / REFERENCES --- p.106 / APPENDIX --- p.118

"Cell and process migration are critical to the establishment of neural circuitry. The study of these processes is facilitated through use of model organisms with simple nervous systems, such as C. elegans. Research in this nematode has defined the cytoplasmic adaptor MIG-10 as a key regulator of these processes. Mutation of mig-10 disrupts neuronal and axonal migration and outgrowth of the ‘canals’, or processes, of the excretory cell. MIG-10 directs the localization of UNC-34, which remodels actin filaments at the leading edge of a migrating cell or process to modify the direction or rate of its protrusion. An interactor of MIG-10 identified in a yeast two- hybrid analysis, ABI-1, has several roles in actin remodeling, such as targeting Ena/VASP members for phosphorylation by Abl kinase. Mutation of abi-1 in the nematode produces phenotypes that resemble those of mig-10 mutants, including disrupted outgrowth of the excretory canals, a developmental process in which ABI-1 is known to function cell autonomously. To test the hypothesis that the ABI-1/MIG-10 interaction contributes to cell migration and outgrowth, both in vivo and in vitro analyses were performed. Expression of either MIG-10A or MIG-10B exclusively in the excretory cell partially rescued the canal truncation characteristic of mig-10 mutants, suggesting MIG-10 functions autonomously in this cell during canal outgrowth. Physical interaction between MIG-10 and ABI-1 was confirmed using a co-immunoprecipitation system. Both MIG-10A and MIG-10B interact with ABI-1 through a mechanism that likely involves the SH3 domain of ABI-1 and sites in either the central region or C-terminus of MIG-10. These results suggest that MIG-10 and ABI-1 function together in a cell autonomous manner to promote cell or process migration. A possible consequence of this interaction is modulation of the MIG-10 binding to UNC-34 through Abl-mediated phosphorylation of MIG-10."

In addition to providing heat insulation and mechanical cushioning, adipose tissue regulates overall metabolic homeostasis and serves as an essential energy storage site. Excess adipose tissue, or obesity, is on the rise in the US among all demographics. The expansion of adipose tissue results from both adipocyte hypertrophy and hyperplasia but the mechanisms that regulate these processes are not fully understood. Destabilizing actin has been shown to promote adipogenesis while actin stabilization inhibits this process. In addition, decreased actin synthesis is known to occur. However, these studies examined total actin and did not consider that actin is in fact a family of functionally diverse isoforms and that individual isoforms may have different functions in adipogenesis. I hypothesized that actin isoforms contribute differently to adipogenic actin reorganization. To measure this, I developed a novel fractionation method that allowed for the reliable quantification of actin polymerization. I used this actin fractionation method to identify an early loss in polymerized α-smooth muscle actin (α-SMA) relative to polymerized β-actin and γ-actin and to also rule out a role for the actin severing protein gelsolin in the loss of polymerized actin. Furthermore, I showed that the loss of α-SMA expression precedes the loss of β-actin and γ-actin expression. A known regulator of actin cytoskeleton genes is the transcription factor serum response factor (SRF) and its co-activator, myocardin related transcription factor (MRTF). I identified a role for MRTF/SRF in the downregulation of actin expression during adipogenesis, particularly α-SMA. There was an additional cAMP-responsive decrease in α-SMA expression during the initiation of adipogenesis by exposure to established inducers. Overall, my findings are consistent with growing evidence suggesting that genetic markers of smooth muscle cells, including α-SMA, help control adipogenic commitment. Understanding these early stages of adipogenesis could open new therapeutic avenues for obesity and its co-morbidities.

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

Actin is a major cytoskeletal protein required for the polarized growth of filamentous fungi. Recent studies have characterized the dynamics of actin polymers in growing Neurospora crassa and identified the presence of actin patches, cables and rings. In Aspergillus nidulans actin patch and ring dynamics have been documented using fluorescent proteins tagged to actin. However, fluorescently tagged actin does not reveal the presence of actin cables. Recently, the Lifeact construct has been used to label all three actin structures in fungi. Lifeact is a 17 amino acid peptide derived from the Saccharomyces cerevisiae actin binding protein Abp140p. To better understand actin dynamics in living cells, A. nidulans was transformed with the Lifeact reporter construct. Lifeact expressing strains grew and developed as wild-type. Lifeact labeled actin localized to three different organizational patterns in mature hyphae: a sub-apical collar of endocytic actin patches that was located approximately 2µm from the apex, an apical actin array, and a sub-apical actin web. The apical actin array (AAA – Apical Actin Array) was present in the apices of forty percent of hyphae observed (n=100). The sub-apical actin web (SAW – Sub-apical Actin Web) was present in fifty percent of hyphae observed and was located at an average of 18.46 µm from the apex. It was hypothesized that this network of actin cables was associated with branch and septation site selection or associated with branch and septa formation. An alternative hypothesis was that the SAW acted as a diffusion barrier for nuclei. It was determined that the SAW was neither associated with branch or septa site selection or formation, nor did it act as a barrier for nuclei. It was observed that the AAA can retract and form the SAW. It was hypothesized that this change in actin dynamics could be connected to the faster growth rates reported for mature hyphae. Measurements of individual hyphae containing the AAA or SAW revealed that hyphae with SAWs grow 1.67 times faster than hyphae with AAAs. This data supports the hypothesis that the presence of the SAW is associated with faster rates of growth. An accumulation of circular vesicles was also observed posterior to the SAW and are believed to be woronin bodies. The identity of the circular structures was not confirmed, but the retraction of the AAA to form the SAW may act as a mechanism to transport apically formed woronin bodies to distal regions of the cell. The SAW may also act as a barrier to maintain woronin bodies in sub-apical regions of the hyphae. The Lifeact actin reporter gave clear and defined labeling of filamentous actin in A. nidulans without disturbing natural development. The use of Lifeact allowed for novel insights into actin cable dynamics present in the apical and sub-apical regions of hyphae, branch formation, and septa formation.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Mar. 23, 2009). Vita. Includes bibliographical references.

Thesis (M. Sc.)--University of Canterbury, 2009. / Typescript (photocopy). "June 2009." Includes bibliographical references (leaves 130-138). Also available via the World Wide Web.