Download Seminars: Molecular and cellular biophysics WS04/05

Séminaires: Biophysique moléculaire et cellulaire II, SS 07
16, 23, 30 avril, 07, 14, 21 mai, 04, 11, 18 juin 2007
Objectif
Apprendre en enseignant. La série de séminaires devrait donner aux étudiants
l’opportunité de présenter des travaux de recherche de pointe concernant les nouvelles
techniques de biophysique utilisées pour étudier des questions centrales en biologie.
Afin de tirer profit des sujets présentés, tous les étudiants devront connaître la
thématique générale en se référant aux livres et articles proposés pendant le cours et
devront avoir lu le résumé donné par l’intervenant ainsi que les principales références.
Général
Chaque étudiant devra donner un séminaire de 20 minutes sur l’un des sujets de son
choix. La présentation sera suivie de 10 minutes de discussion. Pour encourager la
discussion, chaque étudiant devra à la fin de la présentation poser 2 questions générales
sur le sujet de son séminaire.
(De plus amples discussions et questions pourront être posé par e-mail à:
horst.vogel@epfl; [email protected]). Comme plusieurs séminaires sont
regroupés dans une même thématique, les étudiants devront choisir le sujet de leur
présentation de manière à ce que tous les séminaires d’une thématique donnée soient
choisis. Les étudiants dont les séminaires appartiennent au même sujet devront
collaborer afin de présenter une série cohérente de séminaires, où chaque présentation
est la continuation logique de la précédente.
Grandes lignes du séminaire
La présentation devra toujours comprendre (i) une introduction générale de la discipline
ou du sujet traité, (ii) un résumé des résultats, concepts et discussions, (iii) conclusions
principales et messages perçus, et à la fin 2 questions générales pour encourager la
discussion sur le sujet du séminaire.
Chaque étudiant devra délivrer une semaine en avance les figures de sa présentation en
fichier pdf à tous ses collègues et à moi même, ainsi qu’un rapport d’au moins 5 pages
contenant un résumé des grandes lignes du séminaire (points (i)-(iii) mentionnés cidessus) et des informations supplémentaires sur le sujet qui n’ont pas été abordées au
cours du séminaire.
Préparation de la présentation
J’invite chaque groupe d’étudiants à discuter avec moi et mes assistants des différentes
présentations au cours d’une séance de travail de 15 minutes dans mon bureau.
L’objectif de cette réunion est de définir les points importants des sujets qu’il faudra
couvrir dans les séminaires.
Je propose de fixer les dates personnellement par e-mail.
Représentant de classe: s’il vous plaît, préparez une liste des étudiants avec les
séminaires choisis et envoyez la moi par e-mail.
Seminars: Molecular and cellular biophysics II, sem. eté 07
16, 23, 30 April, 07, 14, 21 May, 04, 11, 18 June 2007
Goal
Learning by teaching. The planned series of seminars should give the students the
opportunity to present results of cutting edge research where novel biophysical
techniques are used to investigate central questions in biology. In order to profit from
the subjects presented, all students should know the general topic from the course (use
the topics in the textbooks and in the corresponding scripts proposed in the lecture) and
should have read the summary obtained from the seminar speaker and preferably the
relevant references.
General
Each student should give one seminar of 20 minutes of a topic of his / her own choice.
After the presentation there will be about 5-10 min discussion. To catalyze discussion,
each student should at the end of the seminar pose 2 general questions on the subject of
his/her seminar.
(Further discussions and questions can be posed via e-mail to:
horst.vogel@epfl; [email protected]). Because several seminars are grouped
within one topic-block, students should choose seminar themes always such that all
seminars within a particular block are choosen. If a topic covers several seminars, the
students should collaborate to present a coherent series of seminars, where each seminar
is a logic continuation of the preceeding one.
Seminar outline
During the presentation a seminar should always comprise (i) a general introduction to
the field or to a particular topic, (ii) a summary of results or concepts together with
discussion, (iii) major conclusions & take-home messages, and at the end 2 general
questions to encourage discussion on the subject of the seminar.
Each student should deliver one week in advance his/her figures of the presentation as a
pdf file to all of his/her colleagues and to me together with an additional at least 3-page
report containing a summary of the outline of the seminar (above mentioned points (i)(iii)) and additional information on the subject which were not covered in the seminar
Preparation of the presentation
I would like to invite each (group) of students to discuss with me and my assistants the
planned particular presentation during a tutorial of about 15 mins in my office. The goal
of this tutorial is to define the most important points of the subject which should be
covered in the seminar.
I propose to fix the dates personally by e-mail.
Class representative: Please prepare a list of students with choozen seminars and
send it to me via e-mail.
1.
Lipid rafts
Biological membranes have a very heterogenous composition of lipids and proteins.
Recent findings indicate that cholesterol rich (sub)micrometer lipid domains in cellular
membranes are of central importance for the modulation of the activity of particular
membrane proteins. The field of « rafts » is one of the fastest growing research fields in
biology and biophysics at present.
Seminar 1: Overview of biological importance of rafts
(16 April 07)
- Simons K, Ikonen E: Cell biology - How cells handle cholesterol. SCIENCE 290
(2000) 1721-1726.
- van Meer G: Cell biology - The different hues of lipid rafts, SCIENCE 296 (2002)
855.
Seminar 2: Overview of molecular mechanism of rafts
(16 April 07)
- Jacobson K, Mouritsen OG, Anderson RGW: Lipid rafts: at a crossroad between
cell biology and physics NATURE CELL BIOLOGY 9 (1): 7-14 JAN 2007
- Simons K, Vaz WLC: Model systems, lipid rafts, and cell membranes. ANNUAL
REVIEW BIOPHYSICS BIOMOLECULAR STRUCTURE 33 (2004) 269-295.
2.
Artificial Lipid Systems & Nanotechnology
Seminar 3 : Vesicles as nanoreactors
- Karlsson M, , et al.: Biomimetic nanoscale reactors and networks
ANNUAL REVIEW OF PHYSICAL CHEMISTRY 55: 613-649 2004
(16 April 07)
Seminar 1 : Supported lipid membranes
(23 April 07)
- Motomu Tanaka and Erich Sackmann: Polymer-supported membranes as models of the
cell surface. Nature 437, 656-663 (29 September 2005)
- Groves, J. T. & Boxer, S. G. Micropattern formation in supported lipid membranes.
Acc. Chem. Res. 35, 149−157 ( 2002).
3.
Structure & Function of Membrane Proteins
About 1/3 of the human proteins are membrane proteins. They play central roles in
cellular signaling and belong to the major target proteins of presently used
medicaments. Inspite of their importance for most of the membrane proteins detailed
moleczular structures and functions are missing.
This section concerns structural principles of membrane protein folding and structure,
as well as particular examples of the structure and functions of membrane proteins for
which high resolution structures have been obtained recently.
3.1. Transporters
Membrane transport proteins catalyse the movement of molecules into and out of cells
and organelles which is a central biological reaction to guarantee the proper
functioning of cells. The hydrophobic and metastable nature transport proteins often
makes them difficult to study by traditional means. Novel approaches that have been
developed and applied to several recently crystallized membrane transport proteins.
One of the best investigated example is the lactose permease from Escherichia coli.
The seminars should discuss the general functioning of membrane transport proteins
presenting the broad biophysical approaches and recent crystal structures of different
examples.
Seminar 2: Lactose permease
(23 April 07)
- Abramson J, Kaback HR, Iwata S: Structural comparison of lactose permease and the
glycerol-3-phosphate antiporter: members of the major facilitator superfamily
CURRENT OPINION IN STRUCTURAL BIOLOGY 14 (4): 413-419 AUG 2004
- Guan L, Kaback HR: Lessons from lactose permease
ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE
35: 67-91 2006
Seminar 3: ABC transporters
(23 April 07)
- Kaspar P Locher: Structure and mechanism of ABC transporters
Current Opinion in Structural Biology 14 (2004) Pages 426-431
- Dawson RJP, Locher KP: Structure of a bacterial multidrug ABC transporter
NATURE 443 (7108): 180-185 SEP 14 2006
3.2. Channels
The determination of the structure of several members of the K channel and aquaporin
family represents a unique opportunity to explain the mechanism of these biomolecular
systems. With their ability to go beyond static structures, molecular dynamics
simulations offer a unique route for relating functional properties to membrane channel
structure.
The seminars should present the structure and function of two recently structurally
resolved channels and present experimental and theoretical studies to understand their
molecular function.
Seminar 1: Potassium channels - Structure
(30 April 07)
- MacKinnon R. (Nobel prize 2003): Nobel Lecture. Potassium channels and the atomic
basis of selective ion conduction. Biosci Rep. 2004 Apr;24(2):75-100.
- Long SB, Campbell EB, Mackinnon R: Crystal structure of a mammalian voltagedependent Shaker family K+ channel.
Science. 2005 Aug 5;309(5736):897-903.
- Long SB, Campbell EB, Mackinnon R.: Voltage sensor of Kv1.2: Structural basis of
electromechanical coupling. Science. 2005 Aug 5;309(5736):903-8.
Seminar 2: Potassium channels - Function
(30 April 07)
- Chanda B, Asamoah OK, Blunck R, Roux B, Bezanilla F. Gating charge
displacement in voltage-gated ion channels involves limited transmembrane movement.
Nature. 2005 Aug 11;436(7052):852-6.
- Posson DJ, Ge P, Miller C, Bezanilla F, Selvin PR. Small vertical movement of a K+
channel voltage sensor measured with luminescence energy transfer. Nature. 2005 Aug
11;436(7052):848-51.
Seminar 3: TRP channels
(30 April 07)
- Voets T, Owsianik G, Janssens A, Talavera K, Nilius B. TRPM8 voltage sensor
mutants reveal a mechanism for integrating thermal and chemical stimuli. Nat Chem
Biol. 2007 Mar;3(3):174-82. Epub 2007 Feb 11.
- Voets T, Talavera K, Owsianik G, Nilius B. Sensing with TRP channels. Nat Chem
Biol. 2005 Jul;1(2):85-92. Review.
3.3. G protein-coupled receptors (GPCR): Rhodopsin as a prototypical example
Rhodopsin is a retinal photoreceptor protein of bipartite structure consisting of the
transmembrane protein opsin and a light-sensitive chromophore 11-cisretinal, linked to
opsin via a protonated Schiff base. Studies on rhodopsin have unveiled many structural
and functional features that are common to a large and pharmacologically important
group of proteins from the G protein-coupled receptor (GPCR) superfamily, of which
rhodopsin is the best-studied member. This part will focus on structural features of
rhodopsin as revealed by many biochemical and structural investigations. In particular,
the high-resolution structure of bovine rhodopsin providesa template for understanding
how GPCRs work in general. The seminars should describe (i) the sensitivity and
complexity of rhodopsin that lead to its important role in vision, (ii) how these results
were found by different biophysical techniques and (iii) what can be learned for the
general functioning of GPCRs.
Seminar 1 (2 students, 45 min total)
(07 May 07)
- Filipek S et al.: The crystallographic model of rhodopsin and its use in studies of
other G protein-coupled receptors. ANNUAL REV BIOPHYSICS BIOMOLECULAR
STRUCTURE 32: 375-397 2003
- Hubbell WL, Altenbach C, Hubbell CM, et al.: Rhodopsin structure, dynamics, and
activation: A perspective from crystallography, site-directed spin labeling, sulfhydryl
reactivity, and disulfide cross-linking. Adv Protein Chem. 2003;63:243-90.
- Bulenger S, Marullo S, Bouvier M: Emerging role of homo- and
heterodimerization in G-protein-coupled receptor biosynthesis and maturation
TRENDS IN PHARMACOLOGICAL SCIENCES 26 (3): 131-137 MAR 2005
4
Modern Biophysical Techniques: Recent Applications
4.1 Structural & functional proteomics
Technical advances on several frontiers have expanded the applicability of existing
methods in structural biology and helped close the resolution gaps between them. As a
result, we are now poised to integrate structural information gathered at multiple levels
of the biological hierarchy — from atoms to cells — into a common framework. The
goal is a comprehensive description of the multitude of interactions between molecular
entities, which in turn is a prerequisite for the discovery of general structural principles
that underlie all cellular processes.
Recent successes illustrate the role of mass spectrometry-based proteomics as an
indispensable tool for molecular and cellular biology and for the emerging field
of systems biology. These include the study of protein–protein interactions via
affinity-based isolations on a small and proteome-wide scale, the mapping of
numerous organelles, the concurrent description of the malaria parasite genome
and proteome, and the generation of quantitative protein profiles from diverse
species. The ability of mass spectrometry to identify and, increasingly, to
precisely quantify thousands of proteins from complex samples can be expected to
impact broadly on biology and medicine.
Seminar 2 (2 students, 45 min total):
(07 May 07)
- A Sali, R Glaeser, T Earnest, W Baumeister: From words to literature in structural
proteomics. Nature 422 (2003) 216-225.
- S Renfrey, J Featherstone: Structural proteomics. Nature Reviews Drug Discovery 1
(2002) 175-176.
- R Aebersold, M Mann: Mass spectrometry-based proteomics. Nature 422 (2003) 198207.
4.2 Novel labeling techniques
One of the most important fields of research in biology is the observation and
investigation of complex biochemical reactions in live cells using optical microscopic
techniques. A prerequisite for this approach is the selective in vivo labeling of biomole
with fluorescent reporter groups.
This section concentrates on recent methods in this field.
Seminar 1: Fluorescent Quantum Dots
(14 May 07)
- Bruchez M, et al.: Semiconductor nanocrystals as fluorescent biological labels
SCIENCE 281 (5385): 2013-2016 SEP 25 1998
- Lidke DS et al.: Quantum dot ligands provide new insights into erbB/HER receptormediated signal transduction. NATURE BIOTECHNOLOGY 22 (2): 198-203 FEB
2004
- Jaiswal JK, et al.: Long-term multiple color imaging of live cells using quantum dot
bioconjugates. NATURE BIOTECHNOLOGY 21 (1): 47-51 JAN 2003
Seminar 2: Fusion proteins with fluorescent proteins
(14 May 07)
Verkhusha VV, Lukyanov KA: The molecular properties and applications of Anthozoa
fluorescent proteins and chromoproteins.
NATURE BIOTECHNOLOGY 22 (2004) 289-296.
Seminar 3: Incorporation unnatural amino acids into proteins
(14 May 07)
A Stromgaard, AA Jensen, K Stromgaard: Site-Specific Incorporation of Unnatural
Amino Acids into Proteins. ChemBioChem 5 (2004) 909-916.
4.3 Imaging biochemical networks in live cells
Proteins provide the building blocks for multicomponent molecular units, or pathways,
from which higher cellular functions emerge. These units consist of either assemblies of
physically interacting proteins or dispersed biochemical activities connected by rapidly
diffusing second messengers, metabolic intermediates, ions or other proteins. It will
probably remain within the realm of genetics to identify the ensemble of proteins that
constitute these functional units and to establish the first-order connectivity. The
dynamics of interactions within these protein machines can be assessed in living cells by
the application of fluorescence spectroscopy on a microscopic level, using fluorescent
proteins that are introduced within these functional units. Fluorescence is sensitive,
specific and non-invasive, and the spectroscopic properties of a fluorescent probe can
be analysed to obtain information on its molecular environment. The development and
use of sensors based on the genetically encoded variants of green-fluorescent proteins
has facilitated the observation of ‘live’ biochemistry on a microscopic level, with the
advantage of preserving the cellular context of biochemical connectivity,
compartmentalization and spatial organization. Protein activities and interactions can
be imaged and localized within a single cell, allowing correlation with phenomena such
as the cell cycle, migration and morphogenesis.
Seminar 1
(21 May 07)
- EA Jares-Erijman, TM Jovin: FRET imaging.
NATURE BIOTECHNOLOGY 21 (11): 1387-1395 NOV 2003
- FS Wouters, PJ Verveer, PIH Bastiaens: Imaging biochemistry inside cells
TRENDS in Cell Biology Vol.11 No.5 May 2001
- Lippincott-Schwartz J, Snapp E, Kenworthy A. Studying protein dynamics in living
cells. Nat Rev Mol Cell Biol. 2001 Jun;2(6):444-56. Review.
- Yudushkin IA, Schleifenbaum A, Kinkhabwala A, Neel BG, Schultz C, Bastiaens PI.
Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B.
Science. 2007 Jan 5;315(5808):115-9.
4.4 Single molecule spectroscopy
Recent advances in single-molecule detection and single-molecule spectroscopy at room
temperature by Laser-induced fluorescence offer new tools for the study of individual
macromolecules under physiological conditions. These tools relay conformational
states, conformational dynamics, and activity of single biological molecules to physical
observables, unmasked by ensemble averaging. Distributions and time trajectories of
these observables can therefore be measured during a reaction without the impossible
need to synchronize all the molecules in the ensemble.
Seminar 2
(21 May 07)
- Weiss S: Fluorescence spectroscopy of single biomolecules SCIENCE 283 (1999)
1676-1683.
- Weiss S: Measuring conformational dynamics of biomolecules by single molecule
fluorescence spectroscopy NATURE STRUCTURAL BIOLOGY 7 (9): 724-729 SEP
2000.
- X Michalet et al.: The power and prospects of fluorescence microscopies and
spectroscopies. Annu. Rev. Biophys. Biomol. Struct. 2003. 32:161–82.
Seminar 3:
(21 May 07)
- Hess ST, Huang SH, Heikal AA, et al.: Biological and chemical applications of
fluorescence correlation spectroscopy: A review
BIOCHEMISTRY 41 (3): 697-705 JAN 22 2002
- Sally A Kim and Petra Schwille: Intracellular applications of fluorescence correlation
spectroscopy: prospects for neuroscience
Current Opinion in Neurobiology 2003, 13:583–590
- Bacia K, Kim SA, Schwille P.: Fluorescence cross-correlation spectroscopy in living
cells. Nat Methods. 2006 Feb;3(2):83-9.
4.5 Protein Folding/Unfolding by single molecule techniques
Seminar 1 (2 students, 45 min total):
Protein unfolding by single molecule spectroscopy
- E Rhoades et al: Watching proteins fold one molecule at a time
Proc Natl Acad Sci USA 100 (2003)
(04 June 07)
Protein unfolding by atomic force micro/spectroscopy
- Oesterhelt F et al.: Unfolding pathways of individual bacteriorhodopsins
SCIENCE 288 (5463): 143-146 APR 7 2000.
4.6
Single protein molecule expression in live cells
Seminar 2 (2 students, 45 min total):
(04 June 07)
- Xie XS, Yu J, Yang WY. Living cells as test tubes. Science. 2006 Apr
14;312(5771):228-30.
- Yu J, Xiao J, Ren X, Lao K, Xie XS. Probing gene expression in live cells, one protein
molecule at a time. Science. 2006 Mar 17;311(5767):1600-3.
- Cai L, Friedman N, Xie XS. Stochastic protein expression in individual cells at the
single molecule level. Nature. 2006 Mar 16;440(7082):358-62.
4.7.
Molecular Motors
Seminar 1 (2 students, 45 min total):
(11 June 07)
- Diez M et al.: Proton-powered subunit rotation in single membrane-bound F0F1ATP synthase NATURE STRUCTURAL & MOLECULAR BIOLOGY 11 (2004) 135141.
- Capaldi, R.A. & Aggeler, R.: Mechanism of the F1F0-type ATP synthase, a
biological rotary motor. Trends Biochem. Sci. 27, 154–160 (2002).
- Kinosita K, Adachi K, Itoh H: Rotation of F-1-ATPase: How an ATP-driven
molecular machine may work. ANNUAL REVIEW OF BIOPHYSICS AND
BIOMOLECULAR STRUCTURE 33(2004) 245-268.
4.8
Stochastic Sensing by Single Ion Channels
Seminar 2 (2 students, 45 min total):
(11 June 07)
Principle of stochastic sensing:
- Bayley H, Jayasinghe L: Functional engineered channels and pores - (Review)
MOLECULAR MEMBRANE BIOLOGY 21 (4): 209-220 JUL 2004
- Bayley H, Cremer PS: Stochastic sensors inspired by biology. NATURE 413 (6852):
226-230 SEP 13 2001
- Gu LQ et al.: Stochastic sensing of organic analytes by a pore-forming protein containing a
molecular adapter. NATURE 398 (6729): 686-690 APR 22 1999
Application to nucleic acids:
- Howorka S et al: Kinetics of duplex formation for individual DNA strands within a
single protein nanopore. PROCEEDINGS OF THE NATIONAL ACADEMY OF
SCIENCES OF THE UNITED STATES OF AMERICA 98 (23): 12996-13001 NOV 6
2001
- Bayley H. Sequencing single molecules of DNA. Curr Opin Chem Biol. 2006
Dec;10(6):628-37. Epub 2006 Nov 20. Review.
- Astier Y, Braha O, Bayley H. Toward single molecule DNA sequencing: direct
identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using
an engineered protein nanopore equipped with a molecular adapter.
J Am Chem Soc. 2006 Feb 8;128(5):1705-10.
4.9. Membrane receptor dynamics at the neuronal synapse
Seminar 1:
(18 June 07)
- Courty S, Bouzigues C, Luccardini C, Ehrensperger MV, Bonneau S, Dahan M.
Tracking individual proteins in living cells using single quantum dot imaging.
Methods Enzymol. 2006;414:211-28.
- Dahan M, Levi S, Luccardini C, Rostaing P, Riveau B, Triller A. Diffusion dynamics
of glycine receptors revealed by single-quantum dot tracking. Science. 2003 Oct
17;302(5644):442-5.
- Tardin C, Cognet L, Bats C, Lounis B, Choquet D. Direct imaging of lateral
movements of AMPA receptors inside synapses. EMBO J. 2003 Sep 15;22(18):4656-65.
Seminar 2:
(18 June 07)
- Charrier C, Ehrensperger MV, Dahan M, Levi S, Triller A. Cytoskeleton regulation of
glycine receptor number at synapses and diffusion in the plasma membrane. J Neurosci.
2006 Aug 16;26(33):8502-11.
- Hanus C, Ehrensperger MV, Triller A. Activity-dependent movements of postsynaptic
scaffolds at inhibitory synapses. J Neurosci. 2006 Apr 26;26(17):4586-95.
- Triller A, Choquet D. Surface trafficking of receptors between synaptic and
extrasynaptic membranes: and yet they do move! Trends Neurosci. 2005 Mar;28(3):1339. Review.