Publications by authors named "Victor W Hsu"

46 Publications

Complications and Revision Rates in Minimally Invasive Robotic-Guided Versus Fluoroscopic-Guided Spinal Fusions: The MIS ReFRESH Prospective Comparative Study.

Spine (Phila Pa 1976) 2021 Apr 5. Epub 2021 Apr 5.

Virginia Spine Institute, Reston, VA National Spine Health Foundation, Reston, VA Tabor Orthopedics, Memphis, TN Lyerly Neurosurgery, Jacksonville, FL Atlantic Brain and Spine, Reston, VA Rothman Institute, Abington, PA Department of Orthopedic Surgery, Florida Hospital, Celebration, FL Central Florida Neurosurgery Institute, Osceola, FL Southeastern Spine Center & Research Institute, Sarasota, FL Department of Neurological Surgery, University of Miami Hospital, Miami, FL.

Study Design: Prospective, multi-center, partially randomized.

Objective: Assess rates of complications, revision surgery, and radiation between Mazor robotic-guidance (RG) and fluoro-guidance (FG).

Summary Of Background Data: MIS ReFRESH is the first study designed to compare RG and FG techniques in adult MIS lumbar fusions.

Methods: Primary endpoints were analyzed at 1 year follow-up. Analysis of variables through Cox logistic regression and a Kaplan-Meier Survival Curve of surgical complications.

Results: 9 sites enrolled 485 patients: 374 (RG arm) and 111 (FG arm). 93.2% of patients had >1 year f/u. There were no differences for gender, Charlson Comorbidity Index, diabetes, or tumor. Mean age of RG patients was 59.0 vs. 62.5 for FG (p = 0.009) and BMI was 31.2 vs. 28.1 (p < 0.001). Percentage of smokers was almost double in the RG (15.2% vs. 7.2%, p = .029). Surgical time was similar (skin-to-skin time/#screws) at 24.9 min RG and 22.9 FG (p = 0.550). Fluoroscopy during surgery/#screws was 15.5 sec RG vs. 35.4 sec FG, (15 sec average reduction). Fluoroscopy time during instrumentation/#screws was 3.6 seconds RG vs. 17.8 seconds FG showing an 80% average reduction of fluoro time/screw in RG (p < 0.001). Within one year follow-up, there were 39 (10.4%) surgical complications RG vs. 39 (35.1%) FG, and 8 (2.1%) revisions RG vs. 7 (6.3%) FG. Cox regression analysis including age, gender, BMI, CCI and # screws, demonstrated that the Hazard Ratio (HR) for complication was 5.8 times higher FG vs. RG (95% CI: 3.5-9.6, p < 0.001). HR for revision surgery was 11.0 times higher FG vs. RG cases (95% CI 2.9-41.2, p < 0.001).

Conclusions: Mazor robotic-guidance was found to have a 5.8 times lower risk of a surgical complication and 11.0 times lower risk for revision surgery. Surgical time was similar between groups and robotic-guidance reduced fluoro time per screw by 80% (approximately one minute/case).Level of Evidence: 2.
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http://dx.doi.org/10.1097/BRS.0000000000004048DOI Listing
April 2021

Structural insights into membrane remodeling by SNX1.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.
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http://dx.doi.org/10.1073/pnas.2022614118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958236PMC
March 2021

Combined immunodeficiency due to a mutation in the γ1 subunit of the coat protein I complex.

J Clin Invest 2021 Feb;131(3)

Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1K652E mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.
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http://dx.doi.org/10.1172/JCI140494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843234PMC
February 2021

β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

Cell 2020 12 27;183(6):1520-1535.e14. Epub 2020 Oct 27.

Laboratory of Host-Pathogen Dynamics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. Electronic address:

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that β-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of β-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. β-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.
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http://dx.doi.org/10.1016/j.cell.2020.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590812PMC
December 2020

Coordination of Grp1 recruitment mechanisms by its phosphorylation.

Mol Biol Cell 2020 12 7;31(25):2816-2825. Epub 2020 Oct 7.

Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02115.

The action of guanine nucleotide exchange factors (GEFs) on the ADP-ribosylation factor (ARF) family of small GTPases initiates intracellular transport pathways. This role requires ARF GEFs to be recruited from the cytosol to intracellular membrane compartments. An ARF GEF known as General receptor for 3-phosphoinositides 1 (Grp1) is recruited to the plasma membrane through its pleckstrin homology (PH) domain that recognizes phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we find that the phosphorylation of Grp1 induces its PH domain to recognize instead phosphatidylinositol 4-phosphate (PI4P). This phosphorylation also releases an autoinhibitory mechanism that results in the coil-coil (CC) domain of Grp1 engaging two peripheral membrane proteins of the recycling endosome. Because the combination of these actions results in Grp1 being recruited preferentially to the recycling endosome rather than to the plasma membrane, our findings reveal the complexity of recruitment mechanisms that need to be coordinated in localizing an ARF GEF to an intracellular compartment to initiate a transport pathway. Our elucidation is also remarkable for having revealed that phosphoinositide recognition by a PH domain can be switched through its phosphorylation.
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http://dx.doi.org/10.1091/mbc.E20-03-0173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851867PMC
December 2020

The protein kinase Akt acts as a coat adaptor in endocytic recycling.

Nat Cell Biol 2020 08 15;22(8):927-933. Epub 2020 Jun 15.

Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA, USA.

Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.
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http://dx.doi.org/10.1038/s41556-020-0530-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415567PMC
August 2020

ALDH7A1 inhibits the intracellular transport pathways during hypoxia and starvation to promote cellular energy homeostasis.

Nat Commun 2019 09 6;10(1):4068. Epub 2019 Sep 6.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA.

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics.
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http://dx.doi.org/10.1038/s41467-019-11932-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731274PMC
September 2019

The late stage of COPI vesicle fission requires shorter forms of phosphatidic acid and diacylglycerol.

Nat Commun 2019 07 30;10(1):3409. Epub 2019 Jul 30.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, 60 Fenwood Road, Boston, MA, 02115, USA.

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process.
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http://dx.doi.org/10.1038/s41467-019-11324-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667475PMC
July 2019

ACAP1 assembles into an unusual protein lattice for membrane deformation through multiple stages.

PLoS Comput Biol 2019 07 10;15(7):e1007081. Epub 2019 Jul 10.

Department of Materials Science and Engineering, City University of Hong Kong, Hong Kong, China.

Studies on the Bin-Amphiphysin-Rvs (BAR) domain have advanced a fundamental understanding of how proteins deform membrane. We previously showed that a BAR domain in tandem with a Pleckstrin Homology (PH domain) underlies the assembly of ACAP1 (Arfgap with Coil-coil, Ankryin repeat, and PH domain I) into an unusual lattice structure that also uncovers a new paradigm for how a BAR protein deforms membrane. Here, we initially pursued computation-based refinement of the ACAP1 lattice to identify its critical protein contacts. Simulation studies then revealed how ACAP1, which dimerizes into a symmetrical structure in solution, is recruited asymmetrically to the membrane through dynamic behavior. We also pursued electron microscopy (EM)-based structural studies, which shed further insight into the dynamic nature of the ACAP1 lattice assembly. As ACAP1 is an unconventional BAR protein, our findings broaden the understanding of the mechanistic spectrum by which proteins assemble into higher-ordered structures to achieve membrane deformation.
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http://dx.doi.org/10.1371/journal.pcbi.1007081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663034PMC
July 2019

GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis.

Nature 2018 09 12;561(7722):263-267. Epub 2018 Sep 12.

Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.
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http://dx.doi.org/10.1038/s41586-018-0475-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152935PMC
September 2018

Disrupted N-linked glycosylation as a disease mechanism in deficiency of ADA2.

J Allergy Clin Immunol 2018 10 21;142(4):1363-1365.e8. Epub 2018 Jun 21.

Division of Immunology, Boston Children's Hospital, Boston, Mass; Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, Mass.

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http://dx.doi.org/10.1016/j.jaci.2018.05.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175612PMC
October 2018

Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness.

Sci Rep 2017 01 26;7(1). Epub 2017 Jan 26.

Division of Cell Physiology, Department of Physiology and Cell Biology, Kobe University, Graduate School of Medicine, Kobe, 650-0017, Japan.

Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.
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http://dx.doi.org/10.1038/s41598-016-0028-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428335PMC
January 2017

Reconstitution of COPI Vesicle and Tubule Formation.

Methods Mol Biol 2016 ;1496:63-74

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA, 02115, USA.

The Golgi complex plays a central role in the intracellular sorting of proteins. Transport through the Golgi in the anterograde direction has been explained by cisternal maturation, while transport in the retrograde direction is attributed to vesicles formed by the coat protein I (COPI) complex. A more detailed understanding of how COPI acts in Golgi transport is being achieved in recent years, due in large part to a COPI reconstitution system. Through this approach, the mechanistic complexities of COPI vesicle formation are being elucidated. This approach has also uncovered a new mode of anterograde transport through the Golgi, which involves COPI tubules connecting the Golgi cisternae. We describe in this chapter the reconstitution of COPI vesicle and tubule formation from Golgi membrane.
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http://dx.doi.org/10.1007/978-1-4939-6463-5_6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779305PMC
January 2018

Structural characterization of coatomer in its cytosolic state.

Protein Cell 2016 08 29;7(8):586-600. Epub 2016 Jul 29.

National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
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http://dx.doi.org/10.1007/s13238-016-0296-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980336PMC
August 2016

A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair.

J Cell Biol 2016 06;213(6):631-40

Centro de Estudos de Doenças Crónicas, NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.
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http://dx.doi.org/10.1083/jcb.201511093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915190PMC
June 2016

Cdc42 and Cellular Polarity: Emerging Roles at the Golgi.

Trends Cell Biol 2016 Apr 17;26(4):241-248. Epub 2015 Dec 17.

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Cdc42 belongs to the Rho family of small GTPases and plays key roles in cellular events of polarity. This role of Cdc42 has typically been attributed to its function at the plasma membrane. However, Cdc42 also exists at the Golgi complex. Here we summarize major insights that have been gathered in studying the Golgi pool of Cdc42 and propose that Golgi-localized Cdc42 enables the cell to diversify the function of Cdc42, which in some cases represents new roles and in other cases acts to complement the established roles of Cdc42 at the plasma membrane. Studies on how Cdc42 acts at the Golgi also suggest key questions to address in the future.
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http://dx.doi.org/10.1016/j.tcb.2015.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809763PMC
April 2016

An ACAP1 coat complex acting in endocytic recycling.

Methods Cell Biol 2015 11;130:81-99. Epub 2015 Jun 11.

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA.

A key function of coat proteins is the sorting of protein cargoes into intracellular transport pathways. For many years, however, it has been unclear whether this role of coat proteins would apply to pathways of endocytic recycling. This issue has been clarified in recent years through the identification of multiple coat complexes acting in the recycling pathways. Leading this charge have been studies on a coat complex defined by ACAP1 (adenosine diphosphate ribosylation factor GTPase-activating proteins with Coiled-coil, Ankryin repeat and PH domains 1), which acts in the sorting of cargoes at the recycling endosome for their return to the plasma membrane. This chapter describes the methods used to characterize this role of ACAP1.
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http://dx.doi.org/10.1016/bs.mcb.2015.03.019DOI Listing
June 2016

Coordinated regulation of bidirectional COPI transport at the Golgi by CDC42.

Nature 2015 May 6;521(7553):529-32. Epub 2015 May 6.

1] Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA [2] Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

The Golgi complex has a central role in the intracellular sorting of secretory proteins. Anterograde transport through the Golgi has been explained by the movement of Golgi cisternae, known as cisternal maturation. Because this explanation is now appreciated to be incomplete, interest has developed in understanding tubules that connect the Golgi cisternae. Here we show that the coat protein I (COPI) complex sorts anterograde cargoes into these tubules in human cells. Moreover, the small GTPase CDC42 regulates bidirectional Golgi transport by targeting the dual functions of COPI in cargo sorting and carrier formation. CDC42 also directly imparts membrane curvature to promote COPI tubule formation. Our findings further reveal that COPI tubular transport complements cisternal maturation in explaining how anterograde Golgi transport is achieved, and that bidirectional COPI transport is modulated by environmental cues through CDC42.
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http://dx.doi.org/10.1038/nature14457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449304PMC
May 2015

A PH domain in ACAP1 possesses key features of the BAR domain in promoting membrane curvature.

Dev Cell 2014 Oct 2;31(1):73-86. Epub 2014 Oct 2.

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address:

The BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane curvature. In some cases, a BAR domain also possesses an amphipathic helix that inserts into the membrane to induce curvature. ACAP1 (Arfgap with Coil coil, Ankyrin repeat, and PH domain protein 1) contains a BAR domain. Here, we show that this BAR domain can neither bind membrane nor impart curvature, but instead requires a neighboring PH (Pleckstrin Homology) domain to achieve these functions. Specific residues within the PH domain are responsible for both membrane binding and curvature generation. The BAR domain adjacent to the PH domain instead interacts with the BAR domains of neighboring ACAP1 proteins to enable clustering at the membrane. Thus, we have uncovered the molecular basis for an unexpected and unconventional collaboration between PH and BAR domains in membrane bending.
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http://dx.doi.org/10.1016/j.devcel.2014.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198613PMC
October 2014

Meeting report - Arf and Rab family G proteins.

J Cell Sci 2013 Dec;126(Pt 23):5313-6

Department of Cell Biology, University of Virginia Health System, Charlottesville, VA 22908, USA.

A FASEB Summer Research Conference entitled 'Arf and Rab family G proteins' was held in July 2013 at Snowmass Village, Snowmass, Colorado. Arfs and Rabs are two families of GTPases that control membrane trafficking in eukaryotic cells, and increasing evidence indicates that their functions are tightly coordinated. Because many workers in this field have focused on only one family, this meeting was designed to integrate our understanding of the two families. The conference was organized by Elizabeth Sztul (University of Alabama, Birmingham, USA) and Jim Casanova (University of Virginia, Charlottesville, USA), and provided an opportunity for approximately 90 scientists to communicate their work and discuss future directions for the field. The talks highlighted the structural, functional and regulatory properties of Arf and Rab GTPases and the need to develop coordinated approaches to investigate them. Here, we present the major themes that emerged from the meeting.
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http://dx.doi.org/10.1242/jcs.143610DOI Listing
December 2013

Arl13b regulates endocytic recycling traffic.

Proc Natl Acad Sci U S A 2012 Dec 7;109(52):21354-9. Epub 2012 Dec 7.

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Intracellular recycling pathways play critical roles in internalizing membrane and fluid phase cargo and in balancing the inflow and outflow of membrane and cell surface molecules. To identify proteins involved in the regulation of endocytic recycling, we used an shRNA trafficking library and screened for changes in the surface expression of CD1a antigen-presenting molecules that follow an endocytic recycling route. We found that silencing of the ADP-ribosylation factor (Arf)-like small GTPase Arl13b led to a decrease in CD1a surface expression, diminished CD1a function, and delayed CD1a recycling, suggesting that Arl13b is involved in the regulation of endocytic recycling traffic. Arl13b appears to be required for the major route of endocytic trafficking, causing clustering of early endosomes and leading to the accumulation of endocytic cargo. Moreover, Arl13b colocalized with markers of the endocytic recycling pathway followed by CD1a, namely Arf6 and Rab22a. We also detected an interaction between Arl13b and the actin cytoskeleton. Arl13b was previously implicated in cilia formation and function. Our present results indicate a previously unidentified role for Arl13b in endocytic recycling traffic and suggest a link between Arl13b function and the actin cytoskeleton.
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http://dx.doi.org/10.1073/pnas.1218272110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535586PMC
December 2012

Aquaporin 2 promotes cell migration and epithelial morphogenesis.

J Am Soc Nephrol 2012 Sep 2;23(9):1506-17. Epub 2012 Aug 2.

Center for Systems Biology, Program in Membrane Biology and Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Simches Research Center, 185 Cambridge Street, Boston, MA 02114, USA.

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin β1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin β1 by mutating the RGD motif led to reduced endocytosis, retention of integrin β1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin β1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.
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http://dx.doi.org/10.1681/ASN.2012010079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431417PMC
September 2012

Mechanistic insights into regulated cargo binding by ACAP1 protein.

J Biol Chem 2012 Aug 29;287(34):28675-85. Epub 2012 May 29.

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

Coat complexes sort protein cargoes into vesicular transport pathways. An emerging class of coat components has been the GTPase-activating proteins (GAPs) that act on the ADP-ribosylation factor (ARF) family of small GTPases. ACAP1 (ArfGAP with coiled-coil, ankyrin repeat, and PH domains protein 1) is an ARF6 GAP that also acts as a key component of a recently defined clathrin complex for endocytic recycling. Phosphorylation by Akt has been shown to enhance cargo binding by ACAP1 in explaining how integrin recycling is an example of regulated transport. We now shed further mechanistic insights into how this regulation is achieved at the level of cargo binding by ACAP1. We initially defined a critical sequence in the cytoplasmic domain of integrin β1 recognized by ACAP1 and showed that this sequence acts as a recycling sorting signal. We then pursued a combination of structural, modeling, and functional studies, which suggest that phosphorylation of ACAP1 relieves a localized mechanism of autoinhibition in regulating cargo binding. Thus, we have elucidated a key regulatory juncture that controls integrin recycling and also advanced the understanding of how regulated cargo binding can lead to regulated transport.
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http://dx.doi.org/10.1074/jbc.M112.378810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436524PMC
August 2012

Grp1 plays a key role in linking insulin signaling to glut4 recycling.

Dev Cell 2012 Jun 17;22(6):1286-98. Epub 2012 May 17.

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.

The glucose transporter type 4 (glut4) is critical for metabolic homeostasis. Insulin regulates glut4 by modulating its expression on the cell surface. This regulation is mainly achieved by targeting the endocytic recycling of glut4. We identify general receptor for 3-phosphoinositides 1 (Grp1) as a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6) that promotes glut4 vesicle formation. Grp1 also promotes the later steps of glut4 recycling through ARF6. Insulin signaling regulates Grp1 through phosphorylation by Akt. We also find that mutations that mimic constitutive phosphorylation of Grp1 can bypass upstream insulin signaling to induce glut4 recycling. Thus, we have uncovered a major mechanism by which insulin regulates glut4 recycling. Our findings also reveal the complexity by which a single small GTPase in vesicular transport can coordinate its multiple steps to accomplish a round of transport.
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http://dx.doi.org/10.1016/j.devcel.2012.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376201PMC
June 2012

Getting active: protein sorting in endocytic recycling.

Nat Rev Mol Cell Biol 2012 04 13;13(5):323-8. Epub 2012 Apr 13.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachsuetts 02115, USA.

Endocytic recycling returns proteins to the plasma membrane in many physiological contexts. Studies of these events have helped to elucidate fundamental mechanisms that underlie recycling. Recycling was for some time considered to be the exception to a general mechanism of active cargo sorting in multiple intracellular pathways. In recent years, studies have begun to reconcile this seeming disparity and also suggest explanations for why early recycling studies did not detect active sorting. Further articulation of this emerging trend has far-reaching implications for a deeper understanding of many physiological and pathological events that require recycling.
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http://dx.doi.org/10.1038/nrm3332DOI Listing
April 2012

ARF1 and GBF1 generate a PI4P-enriched environment supportive of hepatitis C virus replication.

PLoS One 2012 16;7(2):e32135. Epub 2012 Feb 16.

Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Cellular levels of phosphatidylinositol 4-phosphate (PI4P) have been shown to be upregulated during RNA replication of several viruses, including the HCV replicon model. However, whether PI4P is required in an infectious HCV model remains unknown. Moreover, it is not established whether the host transport machinery is sequestered by the generation of PI4P during HCV infection. Here we found that PI4P was enriched in HCV replication complexes when Huh7.5.1 cells were infected with JFH1. HCV replication was inhibited upon overexpression of the PI4P phosphatase Sac1. The PI4P kinase PI4KIIIβ was also found to be required for HCV replication. Moreover, the vesicular transport proteins ARF1 and GBF1 colocalized with PI4KIIIβ and were both required for HCV replication. During authentic HCV infection, PI4P plays an integral role in virus replication.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032135PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281116PMC
August 2012

COPI acts in both vesicular and tubular transport.

Nat Cell Biol 2011 Jul 3;13(8):996-1003. Epub 2011 Jul 3.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

Intracellular transport occurs through two general types of carrier, either vesicles or tubules. Coat proteins act as the core machinery that initiates vesicle formation, but the counterpart that initiates tubule formation has been unclear. Here, we find that the coat protein I (COPI) complex initially drives the formation of Golgi buds. Subsequently, a set of opposing lipid enzymatic activities determines whether these buds become vesicles or tubules. Lysophosphatidic acid acyltransferase-γ (LPAATγ) promotes COPI vesicle fission for retrograde vesicular transport. In contrast, cytosolic phospholipase A2-α (cPLA2α) inhibits this fission event to induce COPI tubules, which act in anterograde intra-Golgi transport and Golgi ribbon formation. These findings not only advance a molecular understanding of how COPI vesicle fission is achieved, but also provide insight into how COPI acts in intra-Golgi transport and reveal an unexpected mechanistic relationship between vesicular and tubular transport.
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http://dx.doi.org/10.1038/ncb2273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149785PMC
July 2011

Role of ArfGAP1 in COPI vesicle biogenesis.

Authors:
Victor W Hsu

Cell Logist 2011 Mar;1(2):55-56

Brigham and Women's Hospital; Harvard Medical School; Boston, MA USA.

Studies from our group suggest that ArfGAP1 acts not only as an Arf regulator but also as an Arf effector, with both roles promoting COPI vesicle formation. However, others have concluded differently, specifically that ArfGAP1 only acts as an Arf regulator, which involves inhibition of COPI vesicle formation by preventing components of the COPI complex from binding to target membrane. Here, we propose plausible reconciling explanations for this apparent contradiction.
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http://dx.doi.org/10.4161/cl.1.2.15175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116587PMC
March 2011

ARFGAP1 promotes AP-2-dependent endocytosis.

Nat Cell Biol 2011 May 17;13(5):559-67. Epub 2011 Apr 17.

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.
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http://dx.doi.org/10.1038/ncb2221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087831PMC
May 2011

Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration.

J Cell Biol 2011 Apr;193(1):61-70

Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

During cell migration, integrins are redistributed from focal adhesions undergoing disassembly at the cell's trailing edges to new focal adhesions assembling at leading edges. The initial step of integrin redistribution is thought to require clathrin-mediated endocytosis. However, whether clathrin-mediated endocytosis functions in different contexts, such as basal versus stimulated migration, has not been determined. In this paper, we examine the spatial and temporal redistribution of integrins from focal adhesions upon stimulation by growth factors. Four-dimensional confocal live-cell imaging along with functional analysis reveals that surface integrins do not undergo significant endocytosis at ventral focal adhesions upon cell stimulation with the platelet-derived growth factor. Rather, they abruptly redistribute to dorsal circular ruffles, where they are internalized through macropinocytosis. The internalized integrins then transit through recycling endosomal compartments to repopulate newly formed focal adhesions on the ventral surface. These findings explain why integrins have long been observed to redistribute through both surface-based and internal routes and identify a new function for macropinocytosis during growth factor-induced cell migration.
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http://dx.doi.org/10.1083/jcb.201007003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082178PMC
April 2011