· web viewcaveolae are 60-80 nm bulb-like plasma membrane invaginations discovered more than...

Pasteur course

MOLECULAR BIOLOGY OF THE CELL

PROGRAM 2018-2019 JANUARY 7 – FEBRUARY 8, 2019

MOLECULAR BIOLOGY OF THE CELL

2018 - 2019

January, 7 - February, 8(*), 2019(*) exams included

Directors of the course

Chiara ZURZOLOInstitut PasteurParis, France

Roberto BRUZZONEHKU – Pasteur Research Pole

The University of Hong Kong, Hong Kong

Philippe CHAVRIERInstitut Curie – Research Section

Paris, France

Location

Centre d'Enseignement de l'Institut PasteurLectures : Room n°5 - Building 06, ground floor, Module 3Practical works : Pavillon Louis Martin, building 09, 1st floor

28, rue du Docteur Roux75724 Paris Cedex 15

(*) for Non-Master students, the course will end on FEBRUARY 1, 2019 at 6:00PM.

Description of the course

The Molecular Biology of the Cell course is an intensive laboratory and lecture course of five weeks divided into weekly modules, each focusing on a cutting-edge

aspect of cell biology. It is composed of lectures given by internationally renowned scientists, and of two practical sessions organized together with teams from the Curie and the Pasteur Institutes. The main topics of the course extend across the cell biology of infection, cancer, signaling, epigenetics and intracellular trafficking, emphasizing new experimental approaches. The availability of the core Imaging Platform at Institut Pasteur will introduce students to advanced techniques for the dynamic visualization of cells in health and disease.

Participants are selected from Master 2 students of the University of Paris 6, Paris 7 and Paris XI and foreign postgraduate students. The course is intended to be a platform of excellence in which students can meet and closely interact with worldwide top-level scientists to discuss, exchange ideas and establish valuable contacts in the perspective of establishing a network of young cell biologists at an early stage in their careers. Students will be able to understand the importance of basic research and of a broad interdisciplinary approach to improve human health. We also expect to provide orientations and mentoring to help course alumni in their future career.

The 2018-2019 course is subdivided into two modules. The first will review key biological concepts required to understand tissue morphogenesis. Topics of lectures include: mechanical forces and morphogenesis; epithelial organization and apico-basal cell polarity; collective migration and cell-cell adhesion; micropattern and self organisation; mechanotransduction. The practicum will discuss the main parameters regulating the formation and the remodeling of multicellular tubes in vitro, using micropatterns and microfluidics, and in vivo, using the formation of the tracheal system in Drosophila and the morphogenesis of neural tube in Zebrafish embryos. The second module will provide an overview of key biological concepts required to understand processes that regulate the dynamics of proteins and lipids at the plasma membrane. Topics of lectures include: mechanical forces and caveolae-dependent signaling; glycosylation, endocytosis, and intracellular sorting. It will be complemented by a practicum during which students will investigate caveolae dynamics and mechanotransduction and carbohydrate-based mechanisms to build endocytic pits using chemical biology and imaging techniques.

Practicum 1

ALL YOU WANT TO KNOW ABOUT TUBE FORMATION

Tissue morphogenesis is a multiscale problem integrating modification of cell shape, cell migration, cell position swapping, remodeling of the extracellular environment as well as large scale coordination of cell behavior at the tissue level. To illustrate the different approaches that can be used to tackle a standard morphogenesis problem, we will use a combination of model systems to characterize the main parameters regulating the formation and remodeling of

multicellular tubes, a common tissue shape present throughout Metazoans. Three independent examples of tube formation will be studied by every student in subgroups:

1. The formation of a tube in vitro: We will study the parameters affecting the spontaneous formation of tubes in vitro using a combination of endothelial cells and fibroblasts through long term live imaging, micropatterns and microfluidics.

2. The formation of a large multicellular tube in vivo: We will perform live imaging of the neural tube formation in Zebrafish embryos. To characterize the cellular movements and cell deformations associated with neural tube morphogenesis, we will combine confocal microscopy and single cell tracking in wild type embryos and in embryos upon perturbation of the apico-basal polarity through morpholinos. We will also introduce general concepts regarding Zebrafish handling and live imaging of embryos.

3. Remodeling tubes in vivo: We will study the morphogenesis of the tracheal system in Drosophila embryos. Combining fast live imaging, cell tracking and laser perturbation approaches, we will test which type of forces contribute to the elongation of the terminal dorsal branches of the tracheas. General concepts regarding Drosophila development, fly handling as well as live imaging will also be introduced.

During the second week, data collected on those systems will be analyzed quantitatively using various software and custom procedures (Matlab, Fiji, Imaris). The results obtained on each system will be discussed and compared to outline the universal features regulating tube formation/remodeling. This module will illustrate how the combination of several model systems, with different technical advantages and limitations, can help to answer a similar biological question.

Practicum 2

ENDOCYTOSIS, SIGNALING, CAVEOLAE & GLYCOSYLATION

In the first part of this practicum, we will study how cells use caveolae to translate physical stimuli into biochemical signals by mechanotransduction by which information from the cell surface is transmitted to the nucleus, where gene expression is regulated. Mechanotransduction controls multiple cellular aspects including, but not limited to, cell growth, shape, or differentiation. Abnormal cell responses to external and internal mechanical constraints are often associated with human pathologies such as heart diseases, myopathies and cancer. Caveolae are 60-80 nm bulb-like plasma membrane invaginations discovered more than sixty years ago. Caveolae are generated through tight association of caveolin oligomers, its main structural component, and are stabilized by the assembly of cytoplasmic cavins into a coat-like structure around the caveolae bulb. Our laboratory has shown that caveolae can act as mechanosensing and mechanoprotection cellular organelles. Under increase of membrane tension generated by various types of mechaniocal stress such as cell swelling or stretching, caveolae flatten out immediately to provide additional surface area and prevent the rupture of the plasma membrane. The central role of caveolae in cell mechanics has been extended to a large number of cell types. During the first week, we will:

1) monitor the cytosolic release of the EHD2 ATPase from mechanically disassembled caveolae and its subsequent translocation to the nucleus to mediate mechanotransduction.

2) monitor EHD2 SUMOylation, a post translational modification induced by mechanical stress and involved in EHD2 nucleocytoplasmic shuttling. Through the use of microscopy techniques, this practicum aims at illustrating the key role of caveolae in the response to mechanical stress. It will focus on EHD2, which, by combining both mechanosensing and mechanotransducing activities, plays a central role in the mechanical cell response mediated by caveolae.

In the second part of the practicum, we will study how given endocytic pathways may be early signatures for subsequent intracellular compartmentalization of cargo proteins. We will focus on the well-characterized clathrin pathway as well as on a new endocytic process that was recently hypothesized by our lab, termed the Glycolipid-Lectin (GL-Lect) mechanism. In this latter, glycosylated cargoes, secreted galectins, and glycosphingolipids are key components to initiate membrane curvature for subsequent clathrin-independent endocytic carriers’ formation.For this practical course, we will dissect the endocytic behavior of a particular cargo, the adhesion protein α5β1 integrin, depending on its conformational state. Using the chemical BG/SNAP-tag tool, we have recently demonstrated that the non-ligand-bound conformation of β1 integrin undergoes retrograde trafficking from the plasma membrane to the Golgi compartment. Alteration of canonical retrograde transport machinery is strongly linked with a defect in cell adhesion as well as a dramatic loss of persistent cell migration.We will first at all obtain a proof of concept demonstration for the use of the BG/SNAP tool for the study of retrograde trafficking, using the receptor-binding B-subunit of Shiga toxin (STxB) as a model cargo. Its Golgi accumulation will be monitored by microscopy techniques. We will then analyze by biochemical methods the specific retrograde transport of the different conformations of β1 integrin using conformational state-specific antibodies, in different drug treatment conditions (EGF, GENZ, CBD).In the second part of the program, we will focus on endocytosis. Using fluorescence microscopy and imaging tools, we will analyze the contribution of the GL-Lect mechanism to the endocytic uptake of β1 integrin in different conformational states. Together, our program will provide insights on how the entry mode of a cargo represents a signature for its intracellular compartmentalization and biological activity.

MOLECULAR BIOLOGY OF THE CELL 2018-2019

WEEK 1 January 7-11, 2019

MODULE 1: Mechanobiology and MorphogenesisRomain LEVAYER, Daria BONAZZI, Dorian OBINO, Nicolas DRAY, Institut

Pasteur, and Manuel THÉRY, CEA

Monday, January 7

9:00 – 9:30 Introduction & Presentation of the course Roberto BRUZZONE(HKU-Pasteur Research Pole, Hong Kong)

Philippe CHAVRIER(Institut Curie, Paris, France)

Chiara ZURZOLO(Institut Pasteur, Paris, France)

9:30 – 10:00 Administrative issues

& Presentation of the Education Center Education Center (Institut Pasteur, Paris, France)10:00 – 10:30 Photos for badges (front desk, 25 rue du Dr Roux)

10:30 – 11:30 Self-presentation of the Students (5 min max, 2 slides each, background + project)

12:00 – 13:00 BCI departmental seminar (Amphi JACOB): Thomas LECUITControl and self-organisation of tissue morphogenesis (Institut Biologie du

Développement, Marseille, France)

14:30 – 16:30 Shaping a tissue with mechano-chemical Thomas LECUITinstabilities (Institut Biologie du Développement,

Marseille, France)

17:00 – 18:00 Self-presentation of the Students (5 min max, 2 slides each, background + project)

18:30 – 18:30 Presentation of practical sessions and exam

18:30 – 19:30 Welcome party (Cafétéria, ground floor, Teaching center)

Tuesday, January 8

9:30 – 12:00 Mechanics of blastocyst morphogenesis Jean-Leon MAÎTRE (Institut Curie, Paris, France

13:30 – 16:00 Cytoskeleton self-organization Manuel THERY(CEA, Saclay, France)

16:30 – 17:30 Practicum 1 – Day 1General introduction about tube morphogenesis and presentation of the project

18:00 Paper assignments

Wednesday, January 9

9:30 – 12:00 Mechanobiology of collective cell behaviours Benoît LADOUX(Institut Jacques Monod, Paris, France)

13:30 – 16:00 Cell polarity and tissue morphogenesis Antoine GUICHETin Drosophila (Institut Jacques Monod, Paris, France)

16:30 – 17:30 Practicum 1 – Day 2Preparation of the slides for micropattern

Thursday, January 10

Practicum 1 – Day 3Experiments all day

Friday, January 11

Practicum 1 – Day 4Experiments all day


WEEK 2 JANUARY 14-18, 2019

Monday, January 14

Practicum 1 – Day 5 9:00 - 12:00 Experiments – Image acquisition of the cells culture

12:00 – 13:00 BCI departmental seminar (Amphi JACOB): Xavier TREPATMechanobiology of epithelial growth and folding (IBEC, Barcelona, Spain)

14:30 – 16:30 Mechanobiology of epithelial migration, growth Xavier TREPATand folding (IBEC, Barcelona, Spain)

Tuesday, January 15

9:30 – 12:00 Imaging and stressing multicellular spheroids Pierre NASSOYand organoids (Bioimaging & Optofluidics,

Bordeaux, France)

Practicum 1 - Day 6Afternoon Image acquisition of the cell culture


9:00 – 10:30 Deconstructing cell migration Matthieu PIEL(Institut Curie, Paris, France)

11:00 – 12:30 Cell shape, Microtubule asters Nicolas MINCand early embryogenesis (Institut Jacques Monod, Paris, France)

Practicum 1 – Day 7Afternoon Data analysis


9:00 – 10:30 Host-pathogen interactions in the context Guillaume DUMESNILof blood vessels (Institut Pasteur, Paris, France)

11:00 – 12:30 Fine tuning of tissue morphogenesis Romain LEVAYERby cell death: the role of cell (Institut Pasteur, Paris, France)competition in development and disease

Practicum 1 – Day 8

Afternoon Preparation of presentations

Friday, January 18

9:30 – 12:00 Biomechanical signals and cell behavior Stefano PICCOLO(Department of Molecular Medicine,

University of Padua, Italy)

Practicum 1 – Day 9Afternoon Presentations


WEEK 3 JANUARY 21-25, 2019

MODULE 2: Endocytosis, Signaling, Caveolae & GlycosylationLudger JOHANNES and Christophe LAMAZE, Institut Curie, Paris, France

Monday, January 21

9:30 – 11:30 How can individual mesenchymal cells migrate Roberto MAYORcollectivity: the case of the neural crest (UCL, London, UK)

12:00 – 13:00 BCI departmental seminar (Amphi JACOB): Roberto MAYORWhere is the driving force during collective (UCL, London, UK)cell migration?

Practicum 2 – Day 114:30 – Introduction Practicum Christophe LAMAZE

Tuesday, January 22

Practicum 2 – Day 29:30 – 15:00 Experiments

15:30 – 17:30 Control of signal transduction by caveolae Christophe LAMAZEmechanics (Institut Curie, Paris, France)


Practicum 2 – Day 3 9:00 – 12:00 Experiments

13:30 – 15:30 Super resolution microscopy: Insight into caveolae Ivan Robert NABI structure endoplasmic and reticulum nanodomains (The University of British

Columbia, Vancouver, Canada)

Practicum 2 – Day 315:30 Experiments


Practicum 2 – Day 4 9:00 – 12:00 Image acquisition

13:30 - 15:30 Mechanisms and significance of endosomal sorting Pete CULLEN (School of Biochemistry, University of Bristol, UK)

Practicum 2 – Day 4

16:00 Experiments

Friday, January 25

9:30 – 12:00 Glycosylation Regulation of Cellular Richard CUMMINGSSignaling and Cell Adhesion (National Center for Functional Glycomics,

Harvard Medical School, USA)

Practicum 2 – Day 513:00 Data analysis


WEEK 4 JANUARY 28-FEBRUARY 1, 2019

Monday, January 28


12:00 – 13:00 BCI departmental seminar (Amphi JACOB): Johanna IVASKAFilopodia and integrins in cancer invasion (Orion Corporation and University

of Turku, Finland)

14:30 – 16:30 Integrins in cancer progression Johanna IVASKA(Orion Corporation and University of Turku,

Finland)


Tuesday, January 29


13:30 – 15 :30 Glycosphingolipid-dependent and lectin-driven Ludger JOHANNESconstruction of endocytic pits (Institut Curie, Paris, France)




13:30 – 15:30 Cell Biology of Tumor Invasion Philippe CHAVRIER(Institut Curie, Paris, France)

Practicum 2 – Day 816:00 – 18:00 End of experiments, observations and data analysis


9:30 – 12:00 Dynamics of lipid membranes and protein coats Bruno ANTONNY (IPMC, Marseille, France)

Practicum 2 – Day 913:30 – 16:30 Observations and data analysis

16:30 – Preparation of presentations

Friday, February 1

9:30 – 12:00 Endocytosis, signaling and cancer Pier Paolo DI FIORE(Istituto Europeo di Oncologia, Milano, Italy)

Practicum 2 – Day 1014:00 – 17:00 Presentations

17:00 – 18:00 Oral Examination for Non-Master students

* * * * * * * *


WEEK 5 FEBRUARY 4-8, 2019

FINAL EXAMINATION

Monday, February 4 to Tuesday, February 5

Project preparation. The written project has to be submitted to the Course Committee by Tuesday night.

Wednesday, February 6 to Thursday, February 7

Preparation of the oral examination

Friday, February 8

9:00 – 18:00 Oral examination

__________

DETAILED DESCRIPTION OF THE EXAMINATION

Oral examination on Friday 9 February, 2018 (mark on a 1-20 scale, coefficient 1):

Critical analysis of a scientific article and presentation of an imaginary 3-year research project as follow-up of the results of the article.Presentation: 13 minutes; questions: 7 minutes; total duration: 20 minutes

Organization of the oral presentation: The presentation is open to the public Slides (Powerpoint or other supported formats)

The scientific articles will be given to students during the first week of the course. Each student will write a fictional project intended to be a follow-up of the article received and submit a 4/5 page document to the members of the jury no later than Tuesday 9th February at 20:00. This document should include: Summary of the article (max 1 page) Aims and description of the project (max 3 pages including figures if appropriate) References (max 1 page), using the style of a cell biology journal (e.g., JCB, JCS, MBC,

Cell, NCB….)

DESCRIPTION DETAILEE DE L’EXAMEN

Examen oral le vendredi 9 février 2018 (note sur 20, coefficient 1) :

Présentation critique d’un article et discussion d’un projet fictif sur 3 ans découlant de ces résultats.Présentation : 13 minutes ; questions du jury : 7 minute ; durée totale : 20 minutes.

Organisation de la présentation orale : Exposé public de chaque étudiant devant le jury Diapositives (logiciel Powerpoint ou autre format compatible)

Les articles scientifiques seront donnés aux étudiants pendant la première semaine de cours. Le projet fictif est présenté dans un document de 4/5 pages à remettre au jury au plus tard le mardi 9 février à 20h00, comprenant : Résumé de l’article (max 1 page) Objectifs et description du projet (max 3 pages, figures incluses) Bibliographie (max 1 page) selon le style d’un journal type JCB, JCS, MBC, Cell, NCB…

MOLECULAR BIOLOGY OF THE CELL COURSE

2018 - 2019

ADDRESS DETAILS

DIRECTORS OF THE COURSE

Chiara ZURZOLOMembrane Trafficking & Pathogenesis Unit

Institut Pasteur28, rue du Dr Roux

75724 Paris Cedex 15, FranceTel +33 - (0)1 45 68 82 [email protected]

Roberto BRUZZONEHKU - Pasteur Research PoleThe University of Hong Kong

5 Sassoon RoadPokfulam, Hong KongTel +852 - 2831 5522

[email protected]

Philippe CHAVRIERInstitut Curie - Research Section

CNRS UMR 14426, rue d’Ulm

75248 Paris Cedex 05, FranceTel +33 - (0)1 56 24 63 [email protected]

__________

mailto:[email protected]



ADMINISTRATIVE CONTACTSINSTITUT PASTEUR

Education Center Sylvie GARNEROBuilding 06 – Social building, Module 4Tel: +33 (0) 1 40 61 38 [email protected]

Registrar’s office Sylvie MALOTCéline CORBIN Building 13, room St Joseph de ClunyTel: +33 (0)1 40 61 33 62 [email protected]

· web viewcaveolae are 60-80 nm bulb-like plasma membrane invaginations discovered more than...

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