Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Summary

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Abstract

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15–27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Introduction

The goal of this protocol is to provide an imaging method to localize and track single membrane proteins inside live cells. We call this method Tracking and Localization Microscopy (TALM)^1,2. Like Stochastic Optical Reconstruction Microscopy (STORM)³ and Fluorescence Photoactivation Localization Microscopy ((F)PALM)^4,5, TALM is a single molecule-based fluorescence localization technique. However, it is distinct in the way that the mobility of membrane proteins in combination with repetitive imaging of the same labeled molecule a....

Protocol

The following protocol follows the guidelines of the local institution research ethics committee.

1. Methods

1. Cell culture
   1. Cultivate cells, for example HeLa cells (human cervix carcinoma), in a T25 cell culture flask containing 5 mL of growth medium at 37 °C and 5% CO₂.
      NOTE: For imaging, split the cells onto prepared coverslips (see steps 1.3 and 1.4) and keep in imaging medium.
2. Cell transfection
   NOTE: Use cell lines that stably express the tagged proteins whenever possible²⁴ to avoid strong overexpression. For tra....

Results

Multi-color imaging and colocalization analysis can help to determine the sub-organellar localization of proteins. We demonstrated this earlier with the cytosolic phosphatase and tensin homologue, PINK1, that has different sub-mitochondrial locations due to its processing by mitochondrial proteases¹⁷. PINK1 is an important factor guaranteeing mitochondrial functionality^34,35. To determine the localization o.......

Discussion

Here, a technique for dual color single molecule localization of mobile membrane proteins was presented. Following the protocol, membrane proteins are fused to self-labeling proteins that react with the rhodamine dyes TMR and SiR conjugated to their respective substrates. Rhodamine dyes are bright and photostable and thus allow for repetitive imaging¹. For successful performance, several conditions and critical topics have to be kept in mind.

First, it is important to c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M) (HEPES)	Biochrom	#1104E
DC1: Dichroid beam splitter	Chroma	640 dcxr	NC506031
DC2: Polychroic Mirror, beamsplitter	Chroma	zt405/488/561/640rpc	discontinued
Dulbecco´s Phosphate-Buffered Saline (PBS) 1x (w/o Ca & Mg)	Sigma-Aldrich & Co.	#RNBF8311
Earle´s MEM without phenol red, without L-Glutamine and without NaHCO3 containing 1% FBS, 0.1% HEPES, 0.1% NEAA, 0.1% Alanyl-L-Glutamine and 34.78% sodium hydrogen carbonate (NaHCO3 0.75g/l)			Imaging medium
Earle´s minimum essential medium (MEM) with phenol red, containing 1% Fetal Bovine Serum Superior (FBS), 0.1% HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M), and 0.1% non-essential amino acids (NEAA)			Growth medium
EF: Emission filter quadbandpass	AHF analysentechnik	F72-866	Brightline HC 446 nm/523 nm/600 nm/677 nm
EMCCD camera	Andor	Andor iXON 897	EMCCD camera
Emission filter QuadView filter cubes, orange	AHF analysentechnik	F39-637	bandpass 582 - 619 nm
Emission filter QuadView filter cubes, red	Chroma		bandpass 655 - 725 nm (HQ 690/70)
FBS (Fetal bovine serum) superior	Biochrom	S0615
Fluorescent beads: TetraSpeck™ Microspheres, 0.1 µm, fluorescent blue/green/orange/dark red	Thermo Fisher Scientific	T7279	fluorescent microspheres
Glutamine	Biochrom	#0951C
HeLa cells	DSMZ	ACC-57	Cervical carcinoma cells from patient Henrietta Lacks
Hela cells CI::paGFP, stable	Muster et al., PLOSOne 2010
Hela cells CV g::Halo7-Tag, stable	Appelhans et al., NanoLett 2012
Hela cells Tom20::Halo7-Tag, stable	Appelhans et al., NanoLett 2012
Image splitter	Photometrics	Dual-View QV2	image splitter emission
Imaging processing software	ImageJ2 / Fiji	freeware
Immersion Oil - ImmersolTM 518 F (ne = 1.518, ve = 45)	Carl Zeiss Jena GmbH	444960-0000-000
Inverted epifluorescence microscope	Olympus IX-71/73/83
Laser 561 nm, 200 mW	CrystaLaser	CL-561-200	561 nm emission
Laser 642 nm, 140 mW	Omicron	Luxx-642-140	642 nm emission
MATLAB	MathWorks	version R2013a
MEM with Earle's Balanced Salt Solution 2.2 g/L NaHCO3, stable glutamine w/o PR	Biochrom	FG-0385
MEM with Earle's Balanced Salt Solution with 2.2 g/L NaHCO3, stable glutamine, Phenolred	Biochrom	FG-0325
MitoTracker® Deep Red FM	Thermo Fisher Scientific	M22426	dye
MitoTracker® Green FM	Thermo Fisher Scientific	M7514	dye
Multi-mode-optical polarization maintaining monomode fiber	Pointsource/Qioptiq		KineFLEX
NHS-PEG-MAL, Rapp Polymer	Rapp Polymere GmbH Tübingen		coverslip coating
non-essential amino acids (NEAA)	Biochrom	#0802E
PEG 800 (Polyethylene glycol) 10 %	Carl Roth GmbH	Art No. 0263.1	coverslip coating
Penicillin/Streptomycin	Biochrom	#0122E
Plasmid for PINK1-Halo7-Tag expression	Beinlich et al., ACS Chemical Biology 2015
Poly-L-lysine (1.2 mg/ml)	Sigma-Aldrich & Co.	Cat. No.P9155	coverslip coating
RGD Peptide (Ac-CGRGDS-COOH)	Coring System Diagnostix GmbH, Gernsheim		coverslip coating / Intergrin receptor motif
Silicon Rhodamine linked to HaloTag®-Ligand (SiRHTL)	personal gift from Kai Johnson		dye
Software analysis plugin	self-written C. P. Richter, Biophysik Osnabrück	SLIMFAST 16g
Tetramethylrhodamine / SNAP-Cell® TMR-Star linked to SNAP-Ligand (TMRstar)	New England Biolab®	S9105S	dye
Tetramethylrhodamine linked to HaloTag®-Ligand (TMRHTL)	Promega	G8251	dye
TIRF condensor	Olympus	Cell^TIRF MITICO System	TIRF condensor
TIRF microscope controlling software	Olympus cellSens 1.12
TIRF objective	Olympus	150x oil objective (N.A. 1.45; Olympus UAPO)
Trypsin/EDTA 10x	Biochrom	#0266
Water H2O 99,5 % Rotipuran® Low organic	Carl Roth GmbH	Art. No. HN57.1