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Lab of Cell Biology
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The Lab of Cell Biology will be transferred to the School of Life Science and Technology, as of Dec. 2017.


Research interest

We are interested in studying fundamental principles that allow precise transport and sorting of proteins within mammalian cells. Current research programs in the lab of cell biology at SIAIS are focused on various aspects of organelle biology and membrane trafficking in mammalian cells. Our interest in Golgi biogenesis and secretory pathway stems from Dr.Jim Rothmans groundbreaking discoveries in the 80s and 90s on vesicular transport and mechanism of membrane fusion, ultimately leading to his Nobel prize in physiology or medicine (2013). Correct sorting and delivery of cargos to target membrane-bound organelles is a vital process that is fundamental to normal cell physiology. Secretion of hormones, neurotransmitters and even antibodies to extracellular space is governed by a complex, yet defined rule, and we are working to further our understanding of this important area of cell biology at the molecular level.



We utilize the power of mass spectrometry to unravel the complexity of protein machinery and membrane micro-domains that facilitate cargo sorting, packaging and transport at the Golgi apparatus. In particular, we have been using in vivo tagging/ biotinylation and proteomics approach (called BioID) that allows identification of all proteins in the vicinity of a protein of interest. In this way, we routinely build up a more complete profile of protein compositions of the membrane micro-domains that tremendously assist in dissecting the overall architecture or dynamics of membrane trafficking machineries at the Golgi. Below is the description of various projects in the lab.



Cell Biology Project


Project #1: Mechanism of KDEL receptor trafficking to the ER and to the Cell surface

During protein synthesis, folding and transport, cellular compartments in the secretory pathway face large flux of membranes and proteins, which requires ways to maintain equilibrium among the membrane-bound compartments. One of the ways that this is accomplished is KDEL receptor-dependent retrieval pathway for ER-resident chaperones that was initially identified in yeast in the late 80s by Munro and Pellham (illustration shown here; Molecular Biology of the Cell, Chapter 13, 6th Edition). This work laid foundation for breakthrough research on Unfolded Protein Response (UPR) pathway that result from massive protein misfolding during cellular stress. Recent studies on KDEL receptor function revealed several unexpected roles, including its potential role as a GPCR-like protein along the early secretory pathway and as one of the cell surface receptors for various growth factors and ER-chaperones. However, whether or how KDEL receptor localization and function is regulated on the cell surface is poorly understood.


To better understand KDEL receptor trafficking, we attempted to obtain more complete KDEL receptor interactome using proximity-based in vivo tagging and proteomics. Through RNAi-dependent screening of candidate KDELR interacting proteins, we identified Acyl-CoA Binding Domain Containing 3 (ACBD3) that appears to function as a lynchpin at the Golgi by directly interacting with KDEL receptor and control its Golgi localization and cell surface expression. We are currently investigating how KDEL receptor trafficking is regulated by Acyl-CoA Binding Domain containing 3 (ACBD3). This novel interaction seems responsible for Golgi localization of KDEL receptor, which then facilitates the receptor to capture ER-chaperones and recycle them to the ER for protein folding. Without such receptor-based mechanism, there would be rapid depletion of ER-chaperones in the ER, leading to unfolded protein response (UPR) and cell death.


In ACBD3 knock-down cells (and in knock-out cells), KDEL receptor was found to mis-localize to the ER, indicating that ACBD3 functions as a lynchpin to keep KDELRs parked at the Golgi until binding of KDEL-containing ligands causes dissociation between these two proteins and allowing their retrograde transport to the ER.







Interestingly, we recently found that ACBD3 KO results in increased cell surface expression of KDELR1, as shown in confocal experiments here using 3xFlag tagged KDEL receptor. Cell surface-localized KDEL receptor is known to serve as one of putative receptors for mesencephalic astrocyte-derived neurotropic factor (MANF), which facilitates regeneration of damaged retina (Neves et al,Science 2016) and insulin-secreting pancreatic b-cells (Lindahl et al, Cell Reports 2016).  We feel that basic cell biology of KDEL receptor trafficking is crucial in our understanding of interplay between a cellular stress response and cellular regeneration.

It is currently unknown how KDEL receptor might travel to the cell surface and whether binding of MANF to KDEL receptor at the PM might generate a cellular signal transduction cascade that leads to cellular regeneration. Nevertheless, there is a few reasons to believe that this could be the case, since KDEL receptor is a GPCR-like protein that atypically binds Gao (one of the well-known Ga-subunits known to function at the PM) at the Golgi and controls material delivery from the Golgi to the PM (Solis et al,Cell,2017). The manuscript describing these findings is currently under review in a prestigious cell biology journal.


For a follow-up study, we are focusing on three different aspects of KDEL receptor biology. The first aims to dissect the multi-protein interaction that controls retention and packaging of KDEL receptor, which involves ACBD3, Arf1, ArfGAP1, Gao and b-COP. Yeast two-hydrid assays are currently being utilized to identify point mutations that abrogates KDEL receptor interaction with each of these binding proteins. The second study aims to characterize the cellular itinerary of KDEL receptor trafficking from the Golgi to the cell surface. We are currently testing a hypothesis that KDEL receptor might escape to the recycling endosomes from the Golgi, leading to bulk transport to the plasma membranes and that this slow leakage of the receptor is significantly increased in ACBD3-depleted cells or under cellular stress conditions. Lastly, we aim to investigate whether KDEL receptor binding to MANF may utilize Gao to transmit cellular signal transduction cascades that lead to cellular regeneration and identify the physiological signaling pathways that are significantly influenced by KDEL Receptor-MANF binding at the plasma membranes.


Project #2: Regulation of Rab-Cascades at the medial-Golgi by ACBD3

The Golgi apparatus is a morphologically and functionally complex organelle, which controls a diverse array of post-translational modification of proteins and lipids as well as protein secretory pathway. Due to a large number of secretory and glycosylated cargos traversing the Golgi, a dynamic, yet highly regulated formation of membrane microdomains is required at the cytosolic face of the Golgi apparatus for Golgi stacking/ribbon formation as well as cargo sequestration, sorting and packaging.

By proximity-based in vivo tagging and proteomics (BioID) of the medial-Golgi stacking protein, Golgin45, we have identified Acyl-CoA Binding Domain Containing 3 (ACBD3) as a novel binding partner. Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we are studying the cisternal adhesion complex using a proteomics approach.


We found that Acyl-CoA Binding Domain Containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45 (as shown in co-immunoprecipitation here). ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Further, ACBD3 recruits TBC1D22, a Rab33b GTPase Activating Protein (GAP), to a large multi-protein complex, containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.   We found that ACBD3 directly binds and recruits Golgin45 and a Rab33b-GAP, TBC1D22, to the Golgi (as shown in figure 6 below).


ACBD3 appears to function as a domain organizer for formation of a large multi-protein complex, containing another Golgi stacking protein GRASP55, Golgin45 and TBC1D22. This multi-protein complex is reminiscent of a classic study describing the observation of distinct intercisternal bridges, found in tannic acid-stained Golgi (Cluett and Brown, J.Cell.Science,1992). This study provide a new perspective on the organization of Golgi cisternal adhesion complex at the medial-Golgi. This work has been published in FEBS letters (Yue et al, FEBS Letter,2017; Editors Choice).

We are currently testing the functional consequences of this multi-protein complex formation. Our preliminary results using FRAP approach (Fluorescent Recovery After Photobleaching) indicate that recruitment of TBC1D22 via direct binding to Golgi-associated ACBD3 plays a crucial role in controlling Rab1-Rab33b-Rab6 cascades through the Golgi apparatus. This study further confirms how membrane identities of the Golgi apparatus is dynamically regulated throughout the Golgi stack, just as in cisternal maturation of yeast Golgi membranes (Matsuura-Tokia et al, Nature, 2006; Losev et al, Nature, 2006) and yet, mammalian cells utilize distinct approach to fine tune the flow of membrane identity along the secretory pathway.


Project #3: Mechanism of organelle size control by Tankyrase-1

Cell and organelle size is tightly coupled to cell growth and division. Mammalian cells have been shown to have diverse mechanisms that keep size of intracellular organelles (i.e., nucleus, ER, lysosomes, etc), surface area of plasma membranes and cell volume to optimal level. Size control is also known to be intimately linked to cell division and cell growth. Core understanding of cell size control has derived from the studies of mTOR pathway that regulates cell growth. However, how size of intracellular organelles keeps up in line with cell growth is incompletely understood.





We had recently found via yeast two-hybrid assays that Tankyrase-1 (TNKS1) is a novel binding partner of Golgin45 and promotes poly ADP-ribosylation and ubiquitination of the Golgi stacking protein. This, in turn, results in proteasome-dependent degradation of Golgin45 in steady state cells and help keep the Golgi size within cell-type specific range. Interestingly, Polo-like kinase 1 (Plk1), which had been known to orchestrate mitotic Golgi disassembly, appears to be involved in regulation of TNKS1 activity. We are currently investigating how Plk1-TNKS1 complex may link the Golgi size control to mitotic cell division by controlling Golgin45 protein level in mammalian cells.



 
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