Biol643.001, Seminar in Cell Biology: Cytoskeletal Structure and Function
First Meeting:
Tuesday, August 19th, GSB 1377, 2:00 pm - 3:15 pm
Meeting Time: Tuesdays & Thursdays, 2:00 pm – 3:150 pm
Dec. 7th)
Meeting Location: Genome Sciences Building (GSB) 1377
(12 pm – 3pm, GSB 1377 on Saturday
Course Hours:3
Overview:
This seminar focuses on molecular mechanisms of cytoskeletal components. The course will examine the
actin cytoskeleton and the microtubule cytoskeleton. A sample of topics include 1) the core building
blocks: actin and tubulin; 2) nucleators: Arp2/3 and gamma tubulin/gamma-TuRC/Augmin; 3) motors:
myosin, kinesin and dynein; 4) regulators: formins and microtubule plus end binding proteins; 5)
destabilizers: KinI and stathmin; and 6) kinetochore-microtubule attachments complexes: the Dam1 and
Ndc80 complexes. Primary literature will be examined, presented and critiqued. Each topic will examine a
molecular/mechanistic that correlates structure with mechanism. Emerging techniques in cell biology and
structure will be discussed including single molecule fluorescent techniques (PALM, FIONA, speckle
microscopy), optical trapping, single particle electron microscopy, x-ray crystallography and small angle
X-ray scattering. The course is intended to familiarize cell biologists with molecular mechanisms and
protein structure, promoting proficiency in viewing, evaluating and presenting structure models using
molecular graphics programs in order to design and implement structure-based experiments. The seminar
aims to develop presentation skills, scientific writing, as well as manuscript evaluation and critique.
Methodology:
As a seminar course, we will examine primary literature. Participation from all members is critical. Each
week, a paper will be presented to the group by one or two assigned members. During the presentation,
the paper will be critiqued as a group. People presenting the paper will present the material via
Powerpoint or equivalent program. Aside from presenting figures from the paper, the presenters may be
asked to show structure figures and movies they have created and rendered using molecular graphics
software. Additional papers assigned for the class will be examined in a round-table format. Be prepared
to discuss the paper and the supplementary material.
In order to familiarize students with manuscript preparation techniques and the practice of reviewing
papers for a journal, we will go over aspects of 1) writing cover letters to the editor for manuscript
submission and 2) how to write a review of a manuscript for an editor. The student will prepare two cover
letters, directed at the editor for two of the papers presented in the course. These cover letters will be
presented to the class prior to going over the paper. At two points in the semester, a list of manuscripts
will be posted. Students should choose one paper from each list and serve as a mock referee. The review
will be due at assigned dates shown below.
Towards the end of the semester, there will be a molecular graphics Picture/Movie competition. Students
are to create both an artistic picture and a movie that highlights and conveys specific scientific information
pertinent to mechanism.
At the final class (Dec 6th), each student will give a mini-presentation on a recently published cytoskeletal
paper that probes molecular/cellular mechanism. Papers should selected by the student and approved by
the instructor by Nov 30th.
Grading:
Participation Throughout Course
Presentations (2 x 15%)
Cover Letters (2 x 2.5%)
Reviews:
1st (Midterm)
2nd (Final Exam)
Picture & Movie Competition
Final Mini Presentation
45%
30%
5%
5%
5%
5%
5%
Text:
No text required, this course examines primary literature.
Exams:
The midterm exam and the final exam will take the form of the editorial reviews outlined above. The
midterm review will be due Tuesday, October 14th and the final exam review will be due on Friday,
December 6th.
Building Blocks
1: Course Overview, Tuesday, August 19
Introduction to Protein Structure Determination
Introduction to the Protein Data Bank, pdb files
Introduction to PyMOL Molecular Graphics Program
2: Tubulin, Thursday, August 21
Nogales E, Wolf SG, Downing KH.
Structure of the alpha beta tubulin dimer by electron crystallography.
Nature. 1998 Jan 8;391(6663):199-203. Erratum in: Nature 1998 May 14;393(6681):191.
Dimitrov A, Quesnoit M, Moutel S, Cantaloube I, Poüs C, Perez F.
Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues.
Science. 2008 Nov 28;322(5906):1353-6. Epub 2008 Oct 16.
3: Actin, Tuesday, August 26
Otterbein LR, Graceffa P, Dominguez R.
The crystal structure of uncomplexed actin in the ADP state.
Science. 2001 Jul 27;293(5530):708-11.
Ponti A, Machacek M, Gupton SL, Waterman-Storer CM, Danuser G.
Two distinct actin networks drive the protrusion of migrating cells.
Science. 2004 Sep 17;305(5691):1782-6.
4: Bacterial Actin, Thursday, August 28
Garner EC, Campbell CS, Weibel DB, Mullins RD.
Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog.
Science. 2007 Mar 2;315(5816):1270-4.
Nucleators, Minus End
5: -TuRC / Patronin, Tuesday, September 2
Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA.
Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.
Nature. 2010 Aug 12;466(7308):879-82
Goodwin SS, Vale RD.
Patronin regulates the microtubule network by protecting microtubule minus ends.
Cell. 2010 Oct 15;143(2):263-74. doi: 10.1016/j.cell.2010.09.022.
6: Centrioles I, Thursday, September 4
Kitagawa D, Vakonakis I, Olieric N, Hilbert M, Keller D, Olieric V, Bortfeld M, Erat MC, Flückiger I, Gönczy
P, Steinmetz MO.
Structural basis of the 9-fold symmetry of centrioles.
Cell. 2011 Feb 4;144(3):364-75.
7: Centrioles II, Tuesday, September 9
Mennella V, Keszthelyi B, McDonald KL, Chhun B, Kan F, Rogers GC, Huang B, Agard DA.
Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for
pericentriolar material organization.
Nat Cell Biol. 2012 Nov;14(11):1159-68. doi: 10.1038/ncb2597. Epub 2012 Oct 21.
8: Arp2/3 I, Thursday, September 11
Robinson RC, Turbedsky K, Kaiser DA, Marchand JB, Higgs HN, Choe S, Pollard TD.
Crystal structure of Arp2/3 complex.
Science. 2001 Nov 23;294(5547):1679-84.
9: Arp2/3 II, Tuesday, September 16
Svitkina TM, Borisy GG.
Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin
filament array in lamellipodia.
J Cell Biol. 1999 May 31;145(5):1009-26.
Tip Proteins
10: MT +Tips, Thursday, September 18
Maurer SP, Fourniol FJ, Bohner G, Moores CA, Surrey T.
EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.
Cell. 2012 Apr 13;149(2):371-82.
11: XMAP215 Tuesday, September 23 (Tentative – Guest Instructor)
Ayaz P, Munyoki S, Geyer EA, Piedra FA, Vu ES, Bromberg R, Otwinowski Z, Grishin NV, Brautigam CA,
Rice LM.
A tethered delivery mechanism explains the catalytic action of a microtubule polymerase.
Elife. 2014 Aug 5:e03069. doi: 10.7554/eLife.03069. [Epub ahead of print]
12: Formins, Thursday, September 25
Otomo T, Tomchick DR, Otomo C, Panchal SC, Machius M, Rosen MK.
Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain.
Nature. 2005 Feb 3;433(7025):488-94.
13: Kinetochore I , Tuesday, September 30
Ciferri C, Pasqualato S, Screpanti E, Varetti G, Santaguida S, Dos Reis G, Maiolica A, Polka J, De Luca
JG, De Wulf P, Salek M, Rappsilber J, Moores CA, Salmon ED, Musacchio A.
Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.
Cell. 2008 May 2;133(3):427-39.
Powers AF, Franck AD, Gestaut DR, Cooper J, Gracyzk B, Wei RR, Wordeman L, Davis TN, Asbury CL.
The Ndc80 kinetochore complex forms load-bearing attachments to dynamic microtubule tips via biased
diffusion.
Cell. 2009 Mar 6;136(5):865-75.
14: Kinetochore II, Thursday, October 2
Miranda JJ, De Wulf P, Sorger PK, Harrison SC.
The yeast DASH complex forms closed rings on microtubules.
Nat Struct Mol Biol. 2005 Feb;12(2):138-43.
Aravamudhan P, Felzer-Kim I, Gurunathan K, Joglekar AP.
Assembling the Protein Architecture of the Budding Yeast Kinetochore-Microtubule Attachment using
FRET.
Curr Biol. 2014 Jul 7;24(13):1437-46. doi: 10.1016/j.cub.2014.05.014. Epub 2014 Jun 12.
Destabilizers
15: Spastin & Katanin, Tuesday, October 7
Roll-Mecak A, Vale RD I.
Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin.
Nature. 2008 Jan 17;451(7176):363-7.
16: Spastin & Katanin II, Thursday, October 9
Lindeboom JJ, Nakamura M, Hibbel A, Shundyak K, Gutierrez R, Ketelaar T, Emons AM, Mulder BM,
Kirik V, Ehrhardt DW.
A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.
Science. 2013 Dec 6;342(6163):1245533. doi: 10.1126/science.1245533. Epub 2013 Nov 7.
17: Stathmin I, Tuesday, October 14
(MIDTERM REVIEW DUE)
Gigant B, Curmi PA, Martin-Barbey C, Charbaut E, Lachkar S, Lebeau L, Siavoshian S, Sobel A,
Knossow M.
The 4 A X-ray structure of a tubulin:stathmin-like domain complex.
Cell. 2000 Sep 15;102(6):809-16.
Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, Knossow M.
Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain.
Nature. 2004 Mar 11;428(6979):198-202.
18: Cofilin I, Tuesday, October 21
Paavilainen VO, Oksanen E, Goldman A, Lappalainen P.
Structure of the actin-depolymerizing factor homology domain in complex with actin.
J Cell Biol. 2008 Jul 14;182(1):51-9.
Galkin VE, Orlova A, Kudryashov DS, Solodukhin A, Reisler E, Schröder GF, Egelman EH.
Remodeling of actin filaments by ADF/cofilin proteins.
Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20568-72. doi: 10.1073/pnas.1110109108. Epub 2011
Dec 7.
19: Cofilin II, Thursday, October 23
Suarez C, Roland J, Boujemaa-Paterski R, Kang H, McCullough BR, Reymann AC, Guérin C, Martiel JL,
De la Cruz EM, Blanchoin L.
Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment
boundaries.
Curr Biol. 2011 May 24;21(10):862-8. doi: 10.1016/j.cub.2011.03.064. Epub 2011 Apr 28.
Motors
20: Kinesin I, Tuesday, October 28
Gigant B, Wang W, Dreier B, Jiang Q, Pecqueur L, Plückthun A, Wang C, Knossow M.
Structure of a kinesin-tubulin complex and implications for kinesin motility.
Nat Struct Mol Biol. 2013 Aug;20(8):1001-7. doi: 10.1038/nsmb.2624. Epub 2013 Jul 21.
Kaan HY, Hackney DD, Kozielski F.
The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition.
Science. 2011 Aug 12;333(6044):883-5
21: Kinesin II, Thursday, October 30
Mori T, Vale RD, Tomishige M.
How kinesin waits between steps.
Nature. 2007 Nov 29;450(7170):750-4. Epub 2007 Nov 14.
22: Dynein I, Tuesday, November 4
Redwine WB, Hernández-López R, Zou S, Huang J, Reck-Peterson SL, Leschziner AE.
Structural basis for microtubule binding and release by dynein.
Science. 2012 Sep 21;337(6101):1532-6.
PMID: 22997337 [PubMed - indexed for MEDLINE]
23: Dynein II, Thursday, November 6
Reck-Peterson SL, Yildiz A, Carter AP, Gennerich A, Zhang N, Vale RD.
Single-molecule analysis of dynein processivity and stepping behavior.
Cell. 2006 Jul 28;126(2):335-48.
24: Dynein III, Tuesday, November 11
Qiu W, Derr ND, Goodman BS, Villa E, Wu D, Shih W, Reck-Peterson SL.
Dynein achieves processive motion using both stochastic and coordinated stepping.
Nat Struct Mol Biol. 2012 Jan 8;19(2):193-200.
25: Myosin I, Thursday, November 13
Ménétrey J, Llinas P, Mukherjea M, Sweeney HL, Houdusse A.
The structural basis for the large powerstroke of myosin VI.
Cell. 2007 Oct 19;131(2):300-8.
26: Myosin II, Tuesday, November 18
Kodera N, Yamamoto D, Ishikawa R, Ando T.
Video imaging of walking myosin V by high-speed atomic force microscopy.
Nature. 2010 Nov 4;468(7320):72-6.
27: Motor Wars I, Thursday, November 20
Derr ND, Goodman BS, Jungmann R, Leschziner AE, Shih WM, Reck-Peterson SL.
Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold.
Science. 2012 Nov 2;338(6107):662-5. doi: 10.1126/science.1226734. Epub 2012 Oct 11.
28: Motor Wars II, Tuesday, November 25
Shih SM, Engel BD, Kocabas F, Bilyard T, Gennerich A, Marshall WF, Yildiz A.
Intraflagellar transport drives flagellar surface motility.
Elife. 2013 Jun 11;2:e00744. doi: 10.7554/eLife.00744.
29: Motor Wars III, Tuesday, December 2 (Note: optional KRISTEN VERHEY SEMINAR @ 4:00 PM)
Soppina V, Norris SR, Dizaji AS, Kortus M, Veatch S, Peckham M, Verhey KJ.
Dimerization of mammalian kinesin-3 motors results in superprocessive motion.
Proc Natl Acad Sci U S A. 2014 Apr 15;111(15):5562-7. doi: 10.1073/pnas.1400759111. Epub 2014 Apr
2.
Nakamura M, Chen L, Howes SC, Schindler TD, Nogales E, Bryant Z.
Remote control of myosin and kinesin motors using light-activated gearshifting.
Nat Nanotechnol. 2014 Aug 3. doi: 10.1038/nnano.2014.147. [Epub ahead of print]
30: Special Topics, Saturday, December 6
FINAL REVIEW DUE
Recent Articles – Mini Presentations from all members