Understanding the molecular mechanisms that establish and maintain the Golgi during development, cell division, and cellular stress are significant ongoing goals in biomedical research. Our long-term goal is to identify proteins required for Golgi structure and elucidate their mechanism. This work was initiated using an siRNA-based screen targeting candidates, especially golgins, and cell-based Golgi assembly assays followed by rescue after knockdown in conjunction with biochemical, permeabilized-cell, structural, and computational assays. Our screen identified golgin-160 (G160) as a critical requirement for inward motility of Golgi membranes and showed that its absence arrests Golgi assembly at the dispersed ministack stage impairing directed secretion and cell migration during wound healing. This observation raised the possibility that we could answer fundamental questions concerning the unknown identity and regulation of the motor receptor complex that moves secretory cargo and Golgi membranes inward along microtubules. To test the hypothesis that G160 recruits or activates the dynein motor complex we will 1) identify and functionally characterize its interaction with the motor cytoplasmic dynein, 2) identify and functionally characterize its Arf1-dependent interaction with the Golgi membrane, 3) test the acute dependence of the Golgi on G160-based motility by developing a technique to inactivate G160 using light in a time scale of seconds, and 4) elucidate the control mechanism that allows Golgi dispersal and inheritance during cell division.
These aims are supported by key preliminary findings. G160 was required for microtubule +tip capture of Golgi membranes. The G160 C-term bound directly to the intermediate chain of the dynein complex. G160 was required for dynein Golgi association and it was also sufficient for functional motor recruitment as G160 targeted to mitochondria recruited dynein and induced mitochondrial inward motility. The G160 N-term mediated its Arf1-dependent membrane association and its binding was regulated such that G160 cycled to the cell periphery and returned inward on microtubules. Photo-inactivation of G160 showed that the Golgi acutely depends on G160-based inward motility because early Golgi enzymes constantly cycle to the cell periphery. Finally, G160 dissociated from the Golgi at mitosis but remained bound to dynein and collected at spindle poles. Thus, our experiments are poised to uncover components in the long-sought Golgi receptor for the dynein motor and elucidate the regulation that uncouples Golgi membranes from microtubule-based motility to allow Golgi partitioning into daughter cells.
The goal of this proposal is elucidating the molecular mechanisms in assembly and maintenance of the Golgi apparatus. The Golgi processes newly synthesized proteins and lipids and these reactions are important in preventing and treating human disease. Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. Further, understanding trafficking and its establishment of secretory compartments is a vital concern in the development of therapeutics targeting the multitude of diseases that arise from defective protein products in which these proteins depend on secretory processes such as protein folding, quality control, glycosylation, proteolytic activation, and localization. Such diseases include cystic fibrosis, prion-related diseases, diabetes, and Alzheimer's disease, to name just a few. Human disease also arises from defects in compartment function itself. For example, disorders of glycan synthesis are a substantial and rapidly growing group and it is becoming increasingly evident that the primary defect can be in the transport and localization of the glycan transferees within the membrane trafficking system comprised by the endoplasmic reticulum and the Golgi apparatus.
|Tewari, Ritika; Jarvela, Timothy; Linstedt, Adam D (2014) Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin. Mol Biol Cell 25:3049-58|
|Jarvela, Timothy S; Linstedt, Adam D (2014) The application of KillerRed for acute protein inactivation in living cells. Curr Protoc Cytom 69:12.35.1-12.35.10|
|Song, Lina; Bachert, Collin; Schjoldager, Katrine T et al. (2014) Development of isoform-specific sensors of polypeptide GalNAc-transferase activity. J Biol Chem 289:30556-66|
|Mukhopadhyay, Somshuvra; Linstedt, Adam D (2013) Retrograde trafficking of ABâ‚… toxins: mechanisms to therapeutics. J Mol Med (Berl) 91:1131-41|
|Mukhopadhyay, Somshuvra; Redler, Brendan; Linstedt, Adam D (2013) Shiga toxin-binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms. Mol Biol Cell 24:2311-8|
|Guo, Yusong; Linstedt, Adam D (2013) Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat. Cell Logist 3:e27687|
|Bachert, Collin; Linstedt, Adam D (2013) A sensor of protein O-glycosylation based on sequential processing in the Golgi apparatus. Traffic 14:47-56|
|Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D et al. (2012) Genetically encoded pH sensor for tracking surface proteins through endocytosis. Angew Chem Int Ed Engl 51:4838-42|
|Jarvela, Timothy; Linstedt, Adam D (2012) Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery. J Cell Sci 125:973-80|
|Yadav, Smita; Puthenveedu, Manojkumar A; Linstedt, Adam D (2012) Golgin160 recruits the dynein motor to position the Golgi apparatus. Dev Cell 23:153-65|
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