Cell and Developmental biology

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Intracellular Compartmentation



GROUP LEADER:
Vivek Malhotra (ICREA Research Professor and coordinator of the Cell and Developmental Biology Programme)
STAFF SCIENTIST:
Sandra Mitrovic
POSTDOCTORAL FELLOWS:
Antonios Santos, Cristina Nogeuira,Patrik Erlmann, David Cruz-Garcia, Josse Van Galen, Julien Villeneuve, Juan Duran, Felix Campelo, Amy Curwin, Gerard Cantero, Ishier Raote
PhD STUDENT:
Anupama Ashok
TECHNICIANS:
Margherita Scarpa, Maria Bellido

Summary

Our overall goal is to understand the mechanism of protein secretion and cell compartmentation. The specific goals follow.


Export of PCVII from the ER: A working hypothesis

Our work has led to the identification of a serine threonine kinase called PKD that controls membrane fission to generate transport carriers that are destined to the cell surface. The targets of PKD are mostly proteins that affect lipid composition and sense, or induce, membrane curvature. Our current studies are aimed at reconstituting PKD mediated membrane fission in vitro.

PKD was identified as a protein required for the Ilimaquinone (IQ) mediated vesiculation of the stacked Golgi cisternae. Our new findings reveal the activation of a specific phospholipase by IQ at the Golgi. Chromatography of isolated and detergent solubilized Golgi membranes is in progress to identify this activity for its characterization in membrane fission event.

Genome wide screening approaches has uncovered the involvement of novel proteins in secretion of specific cargoes such as the collagens and mucins. These components, in addition to chemical screens to search for inhibitors that affect mucin secretion are the subject of our on-going efforts.

We identified a new compartment called CUPS and a number of genes required for the secretion of signal sequence lacking proteins secreted by human cells. The mechanism of this unconventional secretion by which proteins such as interleukin (IL)-1, 18 and 37, MIF, tissue transglutaminase, Acyl-CoA binding protein et cetera are secreted, and the dynamics of CUPS is a major focus of the lab.


Highlights

Our work has revealed a novel mechanism by which bulky collagens are exported by addition of membranes, most likely the ERGIC, to a Collagen VII enriched domain of the ER (Nogueira et al., 2014). This concentration of collagen is mediated by a specific protein called TANGO1 that connects collagen in the lumen of the ER to COPII proteins Sec23/Sec24 on the cytoplasmic side of ER exit sites. Upon secretion, collagen VII assembles into long coiled-coiled cables that are necessary for the attachment of the epidermis to the dermis and therefore for skin formation. Patients with mutations in collagen VII gene exhibit blisters in the skin and mucosal membranes (Epidermolysis Bullosa) and there is currently no known cure. Over secretion of Collagen VII produces cylindromas or turban cancer for which there is currently no long term therapy. Our findings are aimed to understand how regulated collagen VII secretion is employed for skin biogenesis and therefore a potential means to search for novel therapeutics for diseases collectively called Epidermolysis Bullosa.

We know little about the mechanism by which proteins lacking a signal sequence for entry into the ER-Golgi pathway are secreted by eukaryotic cells. These non-classical secretory proteins include interleukin 1–ß (a major cell survival factor), tissue transglutaminase (necessary for tissue organization), Diazopam binding inhibitor (that controls sleep and anxiety) and Galectin 3 (which plays a major role in controlling cancer, inflammation, fibrosis and heart disease). We have identified the machinery required for this novel secretory pathway and a new compartment, CUPS, that is generated in the cytoplasm of cells under conditions that promote unconventional protein secretion (Cruz-Garcia et al., 2014). CUPS are made of portions of the ER, Golgi and the endosomes, but lack the enzymes that function in these specific compartments. CUPS are consumed by ER upon shifting cells to normal growth conditions. Thus CUPS form and function only during conditions of nutrient starvation. We want to test the hypothesis that CUPS are a sorting station for unconventional secreted proteins. We want to understand the pathway and mechanism of unconventional mode of secretion.

The biogenesis of vesicles that transport secretory cargoes directly from the trans Golgi network (TGN) to the cell surface is mediates by lipids such as diacylglycerol (DAG) and our new findings have suggested that sphingomyelin levels in the Golgi membranes play a crucial role in this process. When the levels of SM are perturbed, the transmembrane enzyme Sialyltransferase is partitioned from TGN46 (Van Galen et al., 2014). Many proteins work in concert to generate a cargo loaded transport vesicles and SM levels play a critical role in the assembly of these components into a functional domain. This explains the role of SM in vesicle biogenesis at the TGN. Altogether our findings are revealing the significance of DAG production, the role of PKD in generating and controlling the level of SM, and the mechanism by which these components produce a cargo filled transport carrier at the TGN.


Research Projects

  • Mechanism of mucin secretion.
  • Mechanism of collagen export from the ER.
  • Mechanism of transport carrier formation at the Trans Golgi network
  • Mechanism of unconventional protein secretion.

Selected Publications

Nogueira C, Erlmann P, Villeneuve J, António JM Santos, Martínez-Alonso E, Martínez-Menárguez JA and Malhotra V.
“SLY1 and Syntaxin 18 specify a distinct pathway for Procollagen VII export from the endoplasmic reticulum.”
eLIFE, May 19 (2014).

van Galen J, Campelo F, Martínez-Alonso E, Scarpa M, Martínez-Menárguez JA and Malhotra V.
“Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network.”
J Cell Biol, 206:609-618 (2014).

Cruz-Garcia D, Curwin AJ, Popoff JF, Bruns C, Duran JM and Malhotra V.
“Remodeling of secretory compartments to create CUPS during nutrient starvation.”
J Cell Biol, 207:695-703 (2014).