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Dynamics of Intra-Cellular Organization

Keywords : CLIP-170, microtubules, tubuline, sécrétion, Golgi complex, recombinant antibodies

Group leader : Franck Perez

Read the scientific activity report.

Our work focuses on two aspects of cellular dynamics, with an additional strong investment of the development of new technologies. On the one hand, we study the dynamics and function of the Golgi apparatus, the central organelle of the secretory and retrograde trafficking pathways. On the other hand, we study the regulation of microtubule dynamics, and in particular the role played by proteins from the CLIP-170 family in this control. To this end, we have invested in the development of new approaches and tools, like recombinant antibodies, that we are using both for fundamental and applied projects.

Our approaches are based on imaging of living cells and on quantitative analysis of intracellular trafficking, on microtubule dynamics, and on the use of agents that disrupt normal cellular protein function (siRNA, mutants, molecules). We also recently set-up, in collaboration with Jacques Camonis (Inserm U830), a high throughput screening platform based on cellular imaging.
In the past few years, we have strongly invested in the development of the recombinant antibody approach in the context of cell biology (Figure 1, Nizak et al., 2005).

Fig. 1Fig. 1Selection of antibodies in vitro by Antibody Phage Display
Fragments of antibodies corresponding to the variable antibody regions (VH and VL) are expressed in fusion with the PIII protein on the surface of filamentary phages (M13). Phages are produced from  libraries and selected according to their affinity for an immobilized antigen. The selected clones are recovered in bacteria, then used for a new selection cycle. The entire selection takes between 6 and 15 days (3 cycles).

The library we mostly use is a library of scFv (single chain Fv fragments, Fv fragments whose heavy and light chains are combined in the same molecule), comprising more than 109 different clones, and was obtained from the laboratory Greg Winter (MRC, Cambridge, UK).

1 - Recombinant Antibodies

We have developed new methods for preparing the target antigens without expression and purification in E. coli. We have also developed methods for selecting antibodies against particular conformations of the targets or against post-translational modifications. On the other hand, we have simplified and improved our methods of production of recombinant antibodies.
These approaches are used in cell biology research programs and for projects aimed at the treatment of certain forms of cancer. In collaboration with the team of Sebastian Amigorena, we try to improve the effectiveness of an antibody targeting the surface of many cancer cells. Our team is also interested in selecting antibodies sensitive to conformational changes or to post-translational modifications. These antibodies can be used as intra-cellular antibodies (Intrabodies) to track the dynamics of the endogenous protein. (Nizak et al. 2003a, b). In particular, we selected recently an antibody sensitive to conformational changes of tubulin (see below) and antibodies specifically directed against GTP-linked forms of the small GTPase Rab6. (Figure 2, Nizak et al., 2003).

Fig. 2Fig. 2The recombinant antibody anti-Rab6 AA2 is sensitive to conformation changes. A, B: AA2 interacts directly with immobilized GST-Rab6A when Rab6A is bound to GTPγS but not when bound to GDP, in contrast to the GDI protein used as a control (A). AA2 does not bind Rab11 unlike some effectors such as Rab6IP1 that binds to active forms of Rab6A and Rab11 (B). C : AA2 effectively detects Rab6 mutant proteins locked in an active form (a, b) expressed in transfected cells (outlined). Rab6 proteins locked in an inactive conformation are not detected (c, d). In this case only the Rab6 endogenous protein is detected which allows to analyze the effects induced by the overexpression of negative mutant on the behavior of the endogenous Rab6 proteins. (Nizak et al. 2003a - http://www.sciencemag.org/cgi/content/abstract/sci; 300/5621/984)

2 - Structure, dynamics and function of the Golgi

Since several years, we are interested in the role of the Golgi apparatus in cell organization (Chabin-Brion et al, 2001) and in the functions of proteins located on the Golgi apparatus periphery. These proteins are part of the Golgi matrix and are organized around the complex formed by Giantine/GM130/p115. In particular, we studied the Giantin (Nizak et al. 2003b), Rab1 and Rab6 proteins (Del Nery, 2006; Sannerud, 2006; Miserey-Lenkei, 2006). More recently, in collaboration with Y. Wang (University of Michigan, USA), we undertook the study of GRASP-65, another Golgi matrix protein, more particularly involved in Golgi cisternae staking and in Golgi dynamics during mitosis. In general, we question the mechanisms that provide structural and functional homeostasis of the Golgi apparatus. To this end, we studied its mode of inheritance during cell division (Nizak et al., 2004). We have developed a method to specifically inactivate Golgi complexes in intact cells. It enabled us to propose that the Golgi apparatus is formed from elements belonging to the endoplasmic reticulum but its final maturation, both functionally and structurally, requires retrograde transport from already formed Golgi membranes (Jollivet et al., 2007). We have now begun to develop a generic transport assay to study in detail the activity of the Golgi apparatus.

Fig. 3Fig. 3GM130 and Giantin partition differentially after inactivation of the Golgi apparatus.
The matrix proteins GM130 (green) and Giantin (red) are strongly juxtaposed in control cells (a). After Golgi inactivation and recovery, GM130 labels the immature and newly formed Golgi apparatus while the Giantin co-localizes with the inactivated Golgi apparatus. (image obtained by immuno-fluorescence followed by a deconvolution) (Jollivet et al., 2007)

3 - CLIP170, CLIPR and dynamics of microtubules

Microtubules are polarized and dynamic elements of the cytoskeleton. Their minus end, which is slowly polymerizing, is generally oriented toward the center of the cell attached to microtubule organizing centers, while their rapidly polymerizing plus end is often directed towards the cell periphery. The microtubules are subject to a dynamic instability, stochastically alternating between periods of polymerization and depolymerization separated by catastrophe and rescue events.
We are studying the regulation of the dynamics of microtubules (Marceiller et al., 2005, coll. with Christian Poüs' team, Chatenay-Malabry) and certain types of CLIP (cytoplasmic linker proteins) proteins involved in the regulation of microtubule dynamic instability. We showed some years ago (Perez et al., 1999, Diamnatopoulos, 1999) that CLIP-170 is localized in a dynamic way to microtubule polymerizing plus ends (Figure 4). This study founded the class of proteins called +TIPs (+ends tracking proteins) that display a similar dynamics. More recently, we carried-out in the functional analysis of CLIPR-59, a CLIP-170-related protein involved in membrane trafficking and that seems to negatively regulate the dynamics of microtubules (Perez et al, 2002, Lallemand-Breitenbach et al., 2004).

Fig. 4Fig. 4Intracellular localization of CLIP-170
HeLa cells have been fixed and labelled by by immuno-fluorescence using an antibody directed against CLIP-170. Note the comet-like staining of CLIP-170. This corresponds to the plus ends of polymerizing microtubules.

Finally, we selected an antibody sensitive to tubulin conformational changes. This antibody allowed us to verify the presence of a GTP cap at the end of polymerizing microtubules, and also identified the additional and unexpected presence of tubulin-GTP remnants in oldest parts of the polymer. This led us to propose a new model of microtubule dynamic instability (Figure 5, Dimitrov et al., 2008).

Fig. 5Fig. 5A new model for microtubule dynamic instability.
Microtubules polymerize (P) by incorporating tubulin bound to GTP at their plus end (GTP-tubulin in red). GTP is hydrolyzed soon after the assembly (GDP tubulin in black), creating a protective GTP cap. Hydrolysis defects can occur trapping remnants of tubulin-GTP in the polymer (see t3). When the GTP cap is lost, the microtubule is destabilized, and catastrophe (C) and depolymerization (D) events occurs. When a remnant of GTP is uncovered at the end of a depolymerizing microtubule, it can play the role of a GTP cap and rescue (rescue, R) can occur, allowing the microtubule to resume its polymerization (Dimitrov et al., 2008). http://www.sciencemag.org/cgi/rapidpdf/1165401?ijkey=lSqnyr61PJvr.&keytype=ref&siteid=sci

Key publications

  • Year of publication : 2014

  • Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.