Browsing by Author "Banerjee, Diya"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
- Actin polymerization dynamics at the leading edgeHu, Xiaohua (Virginia Tech, 2012-10-05)Actin-based cell motility plays crucial role throughout the lifetime of an organism. While the dendritic nucleation model explains the initiation and organization of the actin network in lamellipodia, two questions need to be answered. In this study, I reconstructed cellular motility in vitro to investigate how actin filaments are organized to coordinate elongation and attachment to leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel beads or thin glass nanofibers coated with N-WASP WCA domains. During sustained motility, physiological concentrations of Mg²⁺ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg²⁺ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, filament capping, or actin shell formation. Addition of other types of crosslinkers restored both comet tail attachment and particle motility. We propose a model in which polycation-induced filament bundling sustains processive barbed end attachment to the leading edge. I lowered actin, profilin, Arp2/3, and CP concentrations to address the generation of actin filament orientation during the initiation of motility. In the absence of CP, Arp2/3 nucleates barbed ends that grow away from the nanofiber surface and branches remain stably attached to nanofiber. CP addition causes shedding of short branches and barbed end capture by the nanofiber. Barbed end retention by nanofibers is coupled with capping, indicating that WWCA and CP bind simultaneously to barbed ends. In pull-down assays, saturating CP addition only blocks WWCA binding to barbed end by half. Labeled WWCA bound to barbed ends with an affinity of 14 pM and unlabeled WWCA with an affinity of 75 pM. CP addition increased WWCA binding slightly at low CP concentrations and decreased WWCA binding to 50% at high CP concentrations. Molecular models of CP and WH2 domains bound respectively to the terminal and penultimate actin subunit showed no overlap and that CP orientation might blocks WWCA dissociation from the penultimate subunit. Simultaneous binding of CP and WWCA to barbed ends is essential to the establishment of filament orientation at the leading edge.
- Characterization of lin-42/period transcriptional regulation by the Ikaros/hunchback-family transcription factor ZTF-16 in Caenorhabditis elegansMeisel, Kacey Danielle (Virginia Tech, 2013-06-03)The gene lin-42 is an ortholog of the mammalian period gene, a component of the circadian pathway that converts environmental stimuli into behavioral and physiological outputs over 24 hours. Mammalian period also regulates adult stem cell differentiation, although this function is poorly understood. The structure, function and expression of lin-42 are all similar to period. Therefore, we are studying lin-42 regulation and function during C. elegans larval development as a model for understanding period control of mammalian stem/progenitor cell development. Previous work has shown that ZTF-16 is a regulator of lin-42 transcription. The lin-42 locus encodes three isoforms, and we have characterized lin-42 isoform specific regulation by ZTF-16 through phenotypic assays and analysis of transcriptional reporter strains. Our data show that ZTF-16 regulates the cyclic expression of lin-42A and lin-42B during larval development. However, ztf-16 is not expressed during the adult stage and does not regulate lin-42C, which is expressed only in adults and may be responsible for the circadian functions of lin-42. We also show that ztf-16 reduction-of-function mutations phenocopy loss-of- function phenotypes of the lin-42A/B isoforms. Finally, we have found that deletion of a putative ZTF-16 transcription factor binding site within the lin-42BC promoter abolishes tissue-specific expression patterns. Together, these data indicate that ZTF-16 is required to regulate the expression of lin-42A/B during C. elegans development, and may do this by direct binding to the lin-42BC promoter. Our findings pave the way for testing the possible regulation of period expression by HIL-family transcription factors in mammalian tissues.
- Characterization of Transcriptional and Post-transcriptional Regulation of lin-42/Period During Post-embryonic Development of C. elegansJames, Tracy (Virginia Tech, 2012-09-11)Period, which is broadly conserved in metazoans, regulates circadian timing of neurophysiology as well as cell fate specification. Studies in mouse and humans indicate that period functions as a tumor suppressor and controls adult stem cell differentiation. However, regulation of period function in developmental pathways has not been characterized and appears to be different from its regulation and function in circadian pathways. lin-42 is the Caenorhabditis elegans ortholog of period and has both circadian and developmental timing functions. During post-embryonic larval development, cyclic expression and function of lin-42 controls stage-specific and reiterative cell fate choices of a subset of epidermal stem cells called seam cells. We are studying lin-42 regulation of seam cell fate during C. elegans larval development as a model for understanding the mechanisms of period regulation of adult stem cell fate in mammals. This dissertation describes the research undertaken to characterize the cis-regulatory elements and the trans-regulatory factors that control lin-42 expression. We used direct molecular interaction assays (Electrophoretic Mobility Shift Assay, EMSA) (Chapter 2) followed by an RNA interference (RNAi)-based genetic screen (Chapter 3) to identify lin-42 transcriptional regulators. Using the EMSA, we identified three 50 to 100 base pair regions (binding regions, BR1-3) in the lin-42 5â noncoding sequences that were bound with specificity by C. elegans nuclear proteins. These binding regions represent putative cis-regulatory elements that may serve as transcription factor binding sites (TFBSs). We attempted to identify by mass spectrometry the proteins that bind to the BR sequences. We also used Phylogenetic Footprinting and bioinformatics screens to identify candidate C. elegans transcription factors (TFs) that may bind to putative TFBSs within the BR sequences. Using an RNAi-based screen, we tested the candidate TF genes for potential genetic interactions with lin-42. We identified ZTF-16, a member of the Hunchback/Ikaros zinc-finger transcription factor family, as a potential lin-42 activator and, using quantitative real-time PCR, confirmed that ztf-16 mutation results in down-regulation and loss of cycling expression of lin-42. We further determined that loss of ztf-16 results in seam cell development defects that phenocopy lin-42 loss-of-function, thus validating ZTF-16 as a transcriptional activator of lin-42.
- Control of sex myoblast migration in C. elegansZhang, Sihui (Virginia Tech, 2013-08-01)Cell migration is critical in generating complex animal forms during development; misregulation of migration contributes to pathological conditions such as cancer metastasis. Thanks to its easily traceable cell lineages in a transparent body and a compact genome accessible to a wealth of genetic manipulations, the use of the nematode C. elegans as a model system has greatly advanced our understanding of mechanisms governing cell migration conserved through higher organisms. Among several migration processes in C. elegans, sex myoblast (SM) migration is an attractive system that has a simple and well-defined migratory route along the ventral side from the posterior to the precise center of the gonad. A multitude of guidance mechanisms control SM migration, many of which are likely to be conserved in other migratory processes. Similar to vertebrate systems, C. elegans uses Rho family small GTPases to regulate the engine of cell motility, the actin cytoskeleton, in response to guidance cues. The differential utilizations of Rho GTPases in distinct processes in vivo remain a central question in the study of Rho GTPases. I investigated how Rho GTPases regulate different aspects of SM migration, and found that Cdc-42/CDC42 functions in the anteroposterior migration, whereas MIG-2/RhoG and CED-10/Rac1 control ventral restriction independently of FGF and SLIT/Robo signaling. The relative difficulty in perturbing SM migration using constitutively active Rho GTPases compared to other migration processes illustrates the robustness of the mechanisms that control SM migration. On a technical aspect, I established a nematode larval cell culture system that allows access to postembryonic cells. Compared to the flourishing genetic researches in C. elegans, there are few studies of molecules that also extend to the subcellular level in postembryonic development, mainly due to the lack of a larval cell culture system. I developed a novel method combining SDS-DTT presensitization of larval cuticles and subsequent pronase E digestion. My method efficiently isolates both low- and high-abundance cell types from all larval stages. This technical advance will not only facilitate studies such as regulation of actin dynamics with high-resolution microscopy, but is beginning to be used by researchers to tackle cell-type specific questions through profiling methods as gene expression analysis.
- Isolation and Culture of Larval Cells from C. elegansZhang, Sihui; Banerjee, Diya; Kuhn, Jeffrey R. (PLOS, 2011-04-29)Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.
- Mathematical modeling approaches for dynamical analysis of protein regulatory networks with applications to the budding yeast cell cycle and the circadian rhythm in cyanobacteriaLaomettachit, Teeraphan (Virginia Tech, 2011-10-24)Mathematical modeling has become increasingly popular as a tool to study regulatory interactions within gene-protein networks. From the modeler's perspective, two challenges arise in the process of building a mathematical model. First, the same regulatory network can be translated into different types of models at different levels of detail, and the modeler must choose an appropriate level to describe the network. Second, realistic regulatory networks are complicated due to the large number of biochemical species and interactions that govern any physiological process. Constructing and validating a realistic mathematical model of such a network can be a difficult and lengthy task. To confront the first challenge, we develop a new modeling approach that classifies components in the networks into three classes of variables, which are described by different rate laws. These three classes serve as "building blocks" that can be connected to build a complex regulatory network. We show that our approach combines the best features of different types of models, and we demonstrate its utility by applying it to the budding yeast cell cycle. To confront the second challenge, modelers have developed rule-based modeling as a framework to build complex mathematical models. In this approach, the modeler describes a set of rules that instructs the computer to automatically generate all possible chemical reactions in the network. Building a mathematical model using rule-based modeling is not only less time-consuming and error-prone, but also allows modelers to account comprehensively for many different mechanistic details of a molecular regulatory system. We demonstrate the potential of rule-based modeling by applying it to the generation of circadian rhythms in cyanobacteria.
- Molecular analysis of the responses of Caenorhabditis elegans (Bristol N2), Panagrolaimus rigidus (AF36) and Panagrolaimus sp. (PS 1579) (Nematoda) to water stressKlage, Karsten (Virginia Tech, 2008-06-24)This work provides a comparative and genetic analysis of the responses to water stress in desiccation-tolerant and desiccation-sensitive nematodes. Caenorhabditis elegans, a model organism for the study of development, aging, and cell biology was shown to be a desiccation-sensitive organism that survives relative humidities above 40\% for periods of up to seven days. Transcripts from the desiccation-tolerant species Panagrolaimus rigidus AF36 and sp. PS1579, which were expressed uniquely during separate desiccation and osmotic stresses, as well as during recovery from exposure to the dual stresses, were cloned. These sequences were used to search for similarities in the genome sequence data of C. elegans. Putative anhydrobiotic-related transcripts were identified that potentially encode heat shock protein 70, late embryogenic abundant protein, and trehalose-phosphate synthase. Other putative genes that were identified within eight separate libraries encode proteins involved in transcription (histones), protein biosynthesis (ribosomal proteins, elongation factors), protein degradation (ubiquitin, proteases), and transport and cell structure (actin, collagen). Gene ontology analysis of the cloned transcripts revealed that developmental processes are activated during exposure to the stresses as well as during recovery, which may suggest a "rejuvenation" process as a key to survival in Panagrolaimus nematodes. Genes that were up-regulated during desiccation stress in C. elegans were classified as belonging either to an early response (until 12 hours of stress), or to a late response (after 12 hours of stress). The early response was characterized by the up-regulation of a large number of genes encoding mono-oxygenases, which may suggest onset of oxidation stress during desiccation of C. elegans. The late response was characterized by the appearance of transcripts encoding proteins of the immune system, heat shock proteins (protein denaturation), and superoxide dismutases (oxidation damage). Genes in C. elegans that were down-regulated in response to desiccation stress include those encoding proteases and lysozymes (metabolic shutdown). Genes that encode channel proteins (water homeostasis) were found among the transcripts up-regulated during recovery of C. elegans. The up-regulation of gpdh-1 and hmit-1.1, two transcripts linked to hyperosmotic stress, suggest that osmotic stress is experienced by C. elegans. Comparison of these data with those obtained from exposure of C. elegans to a range of other stresses showing that the nematode C. elegans uses specific transcripts for the desiccation response; transcripts that are not induced in other stresses such as heat, anoxia or starvation. In addition, transcripts regulated during desiccation stress of C. elegans were also regulated during dauer formation, which may indicate common stress tolerant mechanisms. Recent studies in mammalian cells and C. elegans have shown that microRNAs are able to degrade and to sequester mRNA especially during stress in so called stress bodies. In this study, C. elegans microRNA knock-outs showed a significant decrease in desiccation stress survival compared to wild type C. elegans which may suggest the importance of microRNAs for stress survival in C. elegans and other organisms.