Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Summary

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Abstract

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

Introduction

Cytoplasmic intermediate filaments (IFs) are 10 nm-diameter homo- or heteropolymers of a cell type specific subset of IF proteins. IFs participate in a large range of cellular functions such as cell motility, proliferation and stress responses. Their key role is highlighted by the fact that more than 90 human diseases are directly caused by mutations in IF proteins; for instance, changes in IF composition accompanies tumour growth and spreading^1,2,3. There is growing evidence that the three cytoskeleton systems work in collaboration to control cellular functions such as cell ....

Protocol

1. Coverslips preparation (Day 1, 30 min)

1. Place the 18-mm nº1.5H (170 µm +/- 5µm) glass coverslips on a plastic rack.
2. Put the rack in a 100-mL beaker filled with acetone and soak for 5 min.
3. Transfer the rack to a 100-mL beaker filled with absolute ethanol and soak for 5 min.
4. Transfer the rack to a new 100 mL beaker filled with absolute ethanol and place the beaker in an ultrasonic cleaner device (see Table of materials) at room temperature. Press "on" and wait for 10 min.
5. Put the rack under a laminar flow hood and let the coverslips dry or dry the coverslips with filtered air flow.
6. Pu....

Results

A microscope equipped with 50 mW 405 nm and 100 mW 488, 561 and 642 nm solid-state lasers, an EMCCD 512x512 camera, an alpha Plan Apo 100X/1.46 objective and Band Pass 570-650 / Long Pass 655 emission filters was used for the representative results presented below.

Figure 1A gives an example of molecule density and signal to noise ratio that should be used during raw image acquisition. Good quality .......

Discussion

Critical steps in the protocol

We present here a protocol which minimizes artifacts in the dSTORM images of microtubules and IFs in glial cells. Artifacts can be created at every step of the sample preparation and imaging: fixation, blocking, immunolabeling, drift during acquisition, non-optimal blinking conditions²³. We list below the most critical steps.

Cleaning the coverslips is an important step to limit the non-specific.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Minimum Essential Media (MEM)	Gibco	41090-028	cell culture
non essential amino acid	Gibco	1140-050	cell culture 1/100e
Penicillin-Streptomycin	Gibco	15140-122	cell culture 1/100e
Fœtal Bovine Serum	Gibco	10270-106	cell culture 1/10e
0,05 % Trypsin-EDTA (1X)	Gibco	25300-054	cell culture
PBS 10x	fischer scientific	11540486	cell culture
coverslip 18 mm n°1,5H	Marienfield/Dominic dutsher	900556	coverslips
paraformaldehyde (PFA) solution	Sigma	F8775	cell fixation
glutaraldehyde	Sigma	G5882	cell fixation
triton X100	Sigma	T9284	non ionic surfactant. Used for cell fixation
sodium borohydride (NaBH4)	Sigma	452904-25ML	cell fixation
BSA	Sigma	A7906	sample passivation 3% in PBS
Monoclonal Anti-Vimentin (V9) antibody produced in mouse	Sigma	V6630	primary antibodies
Rat anti alpha tubulin	Biorad	MCA77G	primary antibodies
Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 555	Fisher scientific	10635923	secondary antibodies
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647	Fisher scientific	10226162	secondary antibodies
TetraSpeck Microspheres, 0.1 µm, fluorescent blue/green/orange/dark red	Fisher scientific	T7279	fluorescent beads
Chamlide magnetic chamber	Live Cell Instrument	CM-B18-1	magnetic sample holder, maximum volume 1,2 mL
Cysteamine (MEA)	Sigma	30070	for the blinking buffer
glucose oxidase from Aspergillus niger	Sigma	G0543	for the blinking buffer
glucose	sigma		for the blinking buffer
catalase from bovine liver	Sigma	C9322	for the blinking buffer
Tris (trizma)	Sigma	T1503	for the blinking buffer
Wash-N-Dry coverslip rack	Sigma	Z688568
Parafilm	Sigma	P7793-1EA	paraffin film
ultrasonic cleaner	Fisher scientific	15-335-6
plasma cleaner	Harrick plasma	PDC-32G-2