Expansion microscopy of the mitotic spindle

Ponjavić, Ivana; Vukušić, Kruno; Tolić, Iva M. (2021) Expansion microscopy of the mitotic spindle. In: Methods in Cell Biology. Elsevier, Cambridge, MA, United States, pp. 247-274.

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Abstract

The mitotic spindle is a dynamic and complex cellular structure made of microtubules and associated proteins. Although the general localization of most proteins has been identified, the arrangement of the microtubules in the mitotic spindle and precise localization of various proteins are still under intensive research. However, techniques used previously to decipher such puzzles are resolution limited or require complex microscopy systems. On the other hand, expansion microscopy is a novel super-resolution microscopy technique that uses physical expansion of fixed specimens to allow features closer than the diffraction limit of light (~ 250 nm) to become resolvable in the expanded specimen on a conventional confocal microscope. This chapter focuses on expansion microscopy of the mitotic spindle, specifically using tubulin labeling to visualize all microtubule subpopulations within the spindle. Furthermore, we discuss a protocol for expansion of GFP-tagged proteins, such as protein regulator of cytokinesis 1 (PRC1). We also discuss various approaches for image analysis pointing out main advantages of expansion microscopy when compared to previously used techniques. This approach is currently used in our laboratory to study the architecture of the microtubules in the mitotic spindle after perturbations of various proteins important for the structural and dynamical properties of the mitotic spindle.

Item Type:	Book Section
Uncontrolled Keywords:	Expansion microscopy ; Mitotic spindle ; Microtubules ; Bridging fiber ; Kinetochore fiber ; Tubulin ; PRC1 ; Immunofluorescence
Subjects:	NATURAL SCIENCES > Physics > Biophysics and Medical Physics NATURAL SCIENCES > Biology
Divisions:	Division of Molecular Biology
Projects:	Project title Project leader Project code Project type A new class of microtubules in the spindle exerting forces on kinetochores-NewSpindleForce Iva Tolić 647077 EK Oscilatorna dinamika citoskeleta-OSCITON Iva Tolić IP-2014-09-4753 HRZZ Mehanizmi nastajanja snopova mikrotubula potrebni za sazrijevanje diobenog vretena-SpindleForm Iva Tolić PZS-2019-02-7653 HRZZ Provedba vrhunskih istraživanja u sklopu Znanstvenog centra izvrsnosti za kvantne i kompleksne sustave te reprezentacije Liejevih algebri-QuantiXLie Hrvoje Buljan; Pavle Pandžić KK.01.1.1.01.0004 EK Inovativni protokoli mikroskopije za interdisciplinarna istraživanja u biomedicini-IPSted Iva Tolić KK.01.1.1.04.0057 EK
Depositing User:	Maša Sabo
Date Deposited:	02 Sep 2021 07:35
URI:	http://fulir.irb.hr/id/eprint/6534
DOI:	10.1016/bs.mcb.2020.04.014

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