title: Measuring microtubule polarity in spindles with second-harmonic generation.
abstract: The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation. This method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems.
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts. Electronic address: chehangyu@fas.harvard.edu.
authors.forename: Che-Hang
authors.initials: CH
authors.lastname: Yu
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
authors.forename: Noah
authors.initials: N
authors.lastname: Langowitz
authors.affiliation: Department of Physics, Harvard University, Cambridge, Massachusetts.
authors.forename: Hai-Yin
authors.initials: HY
authors.lastname: Wu
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts; FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts.
authors.forename: Reza
authors.initials: R
authors.lastname: Farhadifar
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts; FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Brugues
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
authors.forename: Tae Yeon
authors.initials: TY
authors.lastname: Yoo
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts; FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts.
authors.forename: Daniel
authors.initials: D
authors.lastname: Needleman
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D002457:Cell Extracts; D016764:Cell Polarity; D003198:Computer Simulation; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts
references.citation: J Neurosci. 2003 Apr 1;23(7):2655-64
references.pmid: 12684451
references.citation: Biophys J. 1986 Oct;50(4):693-712
references.pmid: 3779007
references.citation: J Biomed Opt. 2008 Nov-Dec;13(6):064041
references.pmid: 19123687
references.citation: Biophys J. 2007 May 1;92(9):3251-9
references.pmid: 17293404
references.citation: Nat Protoc. 2006;1(5):2305-14
references.pmid: 17406472
references.citation: Science. 2010 Aug 20;329(5994):967-71
references.pmid: 20724640
references.citation: J Cell Biol. 2004 Feb 2;164(3):347-51
references.pmid: 14757750
references.citation: Opt Express. 2003 Nov 17;11(23):3093-9
references.pmid: 19471431
references.citation: Bioessays. 1989 Jul;11(1):5-9
references.pmid: 2673231
references.citation: Curr Opin Neurobiol. 2004 Oct;14(5):610-6
references.pmid: 15464895
references.citation: Curr Biol. 2004 Sep 21;14(18):R797-805
references.pmid: 15380094
references.citation: Opt Express. 2009 Mar 2;17(5):3679-89
references.pmid: 19259208
references.citation: Cell Cycle. 2010 Mar 15;9(6):1084-90
references.pmid: 20410686
references.citation: Biophys J. 2002 Jan;82(1 Pt 1):493-508
references.pmid: 11751336
references.citation: Biophys J. 2005 Feb;88(2):1377-86
references.pmid: 15533922
references.citation: Dev Cell. 2010 Sep 14;19(3):389-401
references.pmid: 20817616
references.citation: Science. 1998 Jan 23;279(5350):519-26
references.pmid: 9438838
references.citation: J Neurosci. 2010 Sep 29;30(39):12996-3004
references.pmid: 20881117
references.citation: Nat Cell Biol. 2012 May 02;14(5):440-7
references.pmid: 22552143
references.citation: Opt Express. 2009 May 25;17(11):9235-40
references.pmid: 19466174
references.citation: Curr Biol. 2013 Aug 5;23(15):1472-7
references.pmid: 23831293
references.citation: J Biomed Opt. 2008 Nov-Dec;13(6):064039
references.pmid: 19123685
references.citation: Biochem Soc Trans. 2011 Oct;39(5):1149-53
references.pmid: 21936780
references.citation: Opt Lett. 1997 Sep 1;22(17):1323-5
references.pmid: 18188227
references.citation: Methods. 2003 Jan;29(1):97-109
references.pmid: 12543075
references.citation: Annu Rev Cell Dev Biol. 1999;15:141-83
references.pmid: 10611960
references.citation: Cell Motil Cytoskeleton. 1990;16(2):99-103
references.pmid: 2198114
references.citation: Nat Protoc. 2012 Mar 08;7(4):654-69
references.pmid: 22402635
references.citation: Opt Express. 2008 Jul 21;16(15):11574-88
references.pmid: 18648479
references.citation: Cold Spring Harb Protoc. 2010 Dec 01;2010(12):pdb.prot5540
references.pmid: 21123427
references.citation: Opt Express. 2009 Aug 3;17(16):14418-25
references.pmid: 19654849
references.citation: Opt Lett. 2001 Dec 1;26(23):1909-11
references.pmid: 18059734
references.citation: Biophys J. 2006 Jan 15;90(2):693-703
references.pmid: 16258040
references.citation: PLoS Biol. 2010 Oct 19;8(10):e1000516
references.pmid: 20976043
references.citation: Nat Rev Mol Cell Biol. 2008 Nov;9(11):860-73
references.pmid: 18946475
references.citation: Nat Rev Mol Cell Biol. 2009 Jan;10(1):9-20
references.pmid: 19197328
references.citation: Nature. 2000 Sep 7;407(6800):41-7
references.pmid: 10993066
references.citation: Opt Lett. 2001 Apr 15;26(8):485-7
references.pmid: 18040360
references.citation: Dev Neurobiol. 2011 Jun;71(6):403-18
references.pmid: 21557497
references.citation: PLoS One. 2012;7(1):e29795
references.pmid: 22253783
references.citation: Cell. 2012 Apr 27;149(3):554-64
references.pmid: 22541427
references.citation: Nat Cell Biol. 2001 Jan;3(1):E28-34
references.pmid: 11146647
references.citation: Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):15938-43
references.pmid: 15522967
references.citation: Opt Express. 2010 May 10;18(10):10813-21
references.pmid: 20588935
references.citation: J Cell Biol. 1993 Dec;123(6 Pt 1):1475-89
references.pmid: 8253845
references.citation: Chromosoma. 1953;5(5):487-500
references.pmid: 13082662
references.citation: Annu Rev Cell Dev Biol. 1997;13:83-117
references.pmid: 9442869
references.citation: Nature. 1980 Jul 31;286(5772):517-9
references.pmid: 7402333
references.citation: Nat Methods. 2007 Nov;4(11):963-73
references.pmid: 17971781
references.citation: Nature. 1996 Aug 1;382(6590):420-5
references.pmid: 8684481
references.citation: J Cell Biol. 1971 May 1;49(2):468-97
references.pmid: 19866774
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7081-6
references.pmid: 12766225
references.citation: Methods Mol Med. 2007;137:111-23
references.pmid: 18085225
references.citation: J Cell Biol. 1984 Feb;98(2):525-33
references.pmid: 6693493
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11370-5
references.pmid: 18682556
references.citation: J Biomed Opt. 2006 Sep-Oct;11(5):054022
references.pmid: 17092171
references.citation: J Microsc. 2002 Dec;208(Pt 3):190-200
references.pmid: 12460450
references.citation: Int Rev Cytol. 2008;265:111-58
references.pmid: 18275887
references.citation: Open Biol. 2013 Jul 17;3(7):130061
references.pmid: 23864552