Home > Involvement of Cytoplasmic Dynein Light Chains in Apoptotic Process: A Differential Gene Expression Study Using In-Vivo and In-Silico Approaches
Involvement of Cytoplasmic Dynein Light Chains in Apoptotic Process: A Differential Gene Expression Study Using In-Vivo and In-Silico Approaches
Cytoplasmic dynein is a multi-subunit complex that transports cargos along microtubules towards their minus end. Dyneins are very essential for vesicular transport, maintenance of golgi apparatus, spindle formation and cellular homeostasis. They are tightly regulated by the interactions of individual dynein subunits and accessory proteins that are implicated in a wide range of dynein-driven transport events. Malfunctioning of dynein light chains has been associated with a variety of human diseases. Current study is comprised of four projects with the major emphasis on characterization of differentially expressed genes in wild-type verses Tcte3-3 disrupted mice, involved in male germ cell apoptosis. In the first study, basic aim was to explore and evaluate mouse Tcte3 paralogs through Bioinformatics and experimental approaches. Significant changes were observed among Tcte3 paralogs using multiple sequence alignment and restrictions maps analysis. Furthermore, we monitored the expression of Tcte3 paralogs in brain and testis tissues of wild-type and Tcte3-3 disrupted mice. Our findings suggested that observed Tcte3 paralogs exhibited tissue specific expression. Next, we employed two-dimensional gel electrophoresis and quantitative reverse transcriptase PCR approaches and performed detailed in-silico analysis of microarray data extracted from NCBI gene ontology omnibus (GEO) to isolate candidate hits influenced by Tcte3-3 disruption. Our findings elucidated several co-expressed partners of Tcte3 including Anxa5 and Pebp1, whose functional coherence may help in better understanding of apoptotic induction. In another study, to evaluate the gene expression profile of testis and brain tissues, Tcte3-3 knockout mice were characterized by differential gene expression profiling technique using cDNA microarray technology (Illumina Beadchip microarray). Isolated data were analyzed and converted into biologically meaningful form by integrative transcriptomics and proteomics approaches. Furthermore, based on the differential gene expression; gene ontology analysis, pathway analysis and protein-protein interaction mapping analyses were performed. Microarray results were further validated by qRT-PCR, immunoblotting, molecular docking and dynamics simulation analyses. Overall, differential microarray study of Tcte3-3 disrupted mice coupled with detailed Bioinformatics analyses provided an updated information and overall insight into Tcte3 mediated gene regulation thus offering useful clues to counteract or prevent its detrimental consequences. Our results highlighted several differentially expressed genes overrepresented in Gene Ontology categories. Furthermore, molecular interaction and reaction pathways involved in male germ cell apoptosis, cell cycle, lipid metabolism and molecular movement were displayed. As Tcte3 is essential for spermatogenesis, our study revealed valuable resources for the exploration of Tcte3-linked functions in male reproduction. Finally, we analyzed functional conservation of three dynein light chains by exploring their molecular structures to find common interaction networks. We proposed involvement of dynein light chains in apoptotic process via their consistent interactions with common binding partners. Based on this notion, we monitored that Tcte3 and Tctex1 exhibited a similar binding pattern against the previously reported interaction partners of DYNLL1. Taken together, our in-silico and supporting experimental data revealed several novel interaction partners of Tcte3, Tctex1 and Dynll1 with overlapping roles in intrinsic apoptosis pathway. Together, with the exploitation of Tcte3 mediated functions, this study may serve as a valuable resource in understanding complex mechanism of apoptosis. Collectively, our study may contribute towards better understanding of tissue-specific transcriptional control of genes involved in apoptosis by addressing the cooperative influence of diverse regulatory partners, pivotal in unraveling the mechanisms leading to tissue-wise regulation of biological processes.