In vitro assays to study the tracking of shortening microtubule ends and to measure associated forces.
Accurate segregation of mitotic chromosomes relies in part on a strong linkage between the kinetochores and the plus ends of spindle microtubules (MTs). These attachments are maintained even as the MTs shorten from their kinetochore-associated ends, and despite the large variability in the magnitude of load from the chromosomal "cargo." Analysis of the underlying mechanisms has recently been facilitated by the identification and purification of various kinetochore complexes. In this chapter we review some existing approaches to study the interaction of these protein complexes with the ends of shortening MTs in vitro. Specifically, we describe the application of a "segmented" MT technique, which allows quantitative characterization of the tracking of the shortening MT ends by fluorescent proteins and protein-coated beads, as well as controlled measurement of the associated forces. There is a marked similarity between these methods and the approaches that are used to study the motions and forces produced by ATP-dependent motor enzymes walking on coverslip-attached, stable MTs. However, optical resolution at the shortening ends of coverslip-tethered MTs is not as good and the thermal noise is high. Furthermore, there are significant differences in the mechanisms of motions of microbeads driven by motors and by MT depolymerization, as well as in the interpretation of the resulting forces. Clearly, the depolymerization-driven motions are difficult to study and the corresponding phenomenology and theories are more complex than in the motors field. We hope, however, that the relatively straightforward assays based on "segmented" MTs, which are described below, will become a routine methodology, thereby helping to advance the studies of the MT-depolymerization-dependent motility.
Grishchuk
Ekaterina L
EL
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Ataullakhanov
Fazly I
FI
D000465:Algorithms; D000818:Animals; D019411:Clinical Laboratory Techniques / Q000295:instrumentation; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000502:physiology; D055585:Physical Phenomena; D055503:Protein Multimerization / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*
D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins
Cell. 2008 Oct 17;135(2):322-33
18957206
Fungal Genet Biol. 2004 Apr;41(4):411-9
14998524
Methods Cell Biol. 1993;39:149-65
8246795
Mol Biol Cell. 1998 Oct;9(10):2857-71
9763448
Dev Cell. 2009 Mar;16(3):374-85
19289083
EMBO J. 2006 Oct 18;25(20):4888-96
17036054
J Biol Chem. 1994 Mar 18;269(11):8610-5
8132586
Nat Struct Mol Biol. 2007 Aug;14(8):721-6
17643123
Proc Natl Acad Sci U S A. 2005 Mar 22;102(12):4353-8
15767580
Biophys J. 1995 Apr;68(4 Suppl):230S-239S; discussion 239S-241S
7787083
J Cell Biol. 2006 Jan 2;172(1):27-39
16380440
Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19017-22
18029449
Methods Enzymol. 1991;196:478-85
2034137
Nature. 2005 Nov 17;438(7066):384-8
16292315
J Cell Biol. 2007 Jul 16;178(2):269-81
17620411
J Cell Biol. 1988 Sep;107(3):1011-24
3417772
Methods Cell Biol. 1993;39:105-13
8246791
Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15423-8
18824692
Nat Cell Biol. 2007 Jul;9(7):832-7
17572669
Proc Natl Acad Sci U S A. 1985 Jul;82(13):4404-8
3859869
Annu Rev Biochem. 2007;76:563-91
17362199
J Cell Biol. 1995 Jan;128(1-2):117-25
7822409
Cell. 2009 Mar 6;136(5):865-75
19269365
Nature. 2006 Mar 23;440(7083):565-9
16415853
Mol Cell. 2005 Jan 21;17(2):277-90
15664196
J Cell Biol. 1991 Mar;112(6):1165-75
1999468
Nature. 2006 May 4;441(7089):115-9
16672973
Mol Biol Cell. 1997 Apr;8(4):607-20
9247642
Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9873-8
16777964
Biophys J. 1995 Dec;69(6):2268-76
8599634
Nat Rev Mol Cell Biol. 2008 Apr;9(4):309-22
18322465
Annu Rev Cell Dev Biol. 1997;13:83-117
9442869
Biophys J. 1998 Sep;75(3):1529-40
9726955
Nature. 1995 Jan 12;373(6510):161-4
7816099
Mol Biol Cell. 2009 Feb;20(3):963-72
19037096
Proc Natl Acad Sci U S A. 2008 May 13;105(19):6918-23
18460602
J Cell Biol. 1973 Nov;59(2 Pt 1):267-75
4805001
Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5209-13
8506368
Curr Biol. 1994 Dec 1;4(12):1053-61
7704569
Dev Cell. 2008 Nov;15(5):645-55
19000831
Nature. 2006 Aug 10;442(7103):709-12
16799566
Annu Rev Biophys Biomol Struct. 2001;30:397-420
11441808