Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Summary

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

Abstract

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

Introduction

Cell membrane maintains the integrity of cells by providing a barrier between the cell and the extracellular environment. A chemical, electrical, or mechanical stimulus that exceeds the normal physiological threshold as well as the presence of invading pathogens can each result in injury to the cell membrane and trigger a subsequent cellular response to repair this injury. To survive these injuries to the cell membrane, cells possess an efficient mechanism for repair. This mechanism is calcium dependent and involves intracellular trafficking of proteins such as annexins and MG53 amongst others as well as subcellular compartments such as endosomes, lysosomes, Golgi der....

Protocol

1. Imaging Cell Membrane Repair Using Bulk (Glass Bead) Wounding

This protocol allows separately marking the injured cells and those that fail to heal. Quantifying these populations of cells requires use of three conditions: 1. Test (C1) - Cells are allowed to repair in presence of Ca²⁺, 2. Control 1 (no injury C2) - Cells are incubated in presence of Ca²⁺, but not injured, and 3. Control 2 (no repair C3) - cells are allowed to repair in absence of Ca²⁺.

1. Grow cells to >50% confluence on three sterile coverslips and wash C1 and C2 twice with CIM at 37 °C and C3 with PBS at 37 °C and ....

Results

Protocols described here for single cell imaging are to monitor the ability and kinetics of cell membrane repair (Protocols 1 and 2) and the subcellular trafficking and fusion of lysosomes during repair (Protocols 3 and 4).

Protocol 1 shows a bulk assay that allows marking all injured cells and identifying those injured cells that failed to repair. The results in Figure 1 show that while the uninjured cells (Figure 1A) remain unlabeled, cells injured by glass .......

Discussion

Cell membrane injury in vivo occurs due to a variety of physiological stressors and several experimental approaches have been developed to mimic these. These include injuring cell membrane of adherent cells by scraping them off the dish or by passaging through a narrow bore syringe^9,10. Following such injuries the cells heal in suspension and not adhered to the extracellular matrix as they normally do in the tissue. Still others, such as use of pore forming toxins chemically alter cell membrane by ext.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HBSS	Sigma	H1387	Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)
HEPES	Fisher	BP410	Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)
FITC dextran (Lysine fixable)	Invitrogen	D1820	2 mg/ml in CIM or PBS
Glass beads	Sigma	G8772
TRITC dextran (Lysine fixable)	Invitrogen	D1818	2 mg/ml in CIM or PBS
PFA 16%	EMS	15710	4% in PBS
Hoechst	Invitrogen	H3570	1/10 000 in PBS
FM dye	Invitrogen	T3163	1 µg/µl in CIM or PBS
FITC dextran	Sigma	FD40S	2 mg/ml in growth medium
LAMP1	Santa Cruz	sc-19992	1/300 in growth medium
Alexa Fluor 488 chicken anti-rat IgG	Invitrogen	A21470	1/500 in blocking solution
Mounting media	Dako	S3023
Coverslips	Fisher	12-545-86
Glass bottom petridish	MatTek corporation	P35G-1.0-14-C
Silicone O ring	Bellco	1943-33315
Coverslip holder	Bellco	1943-11111
Invitrogen	A-7816
DMEM	VWR	12001-566
FBS	VWR	MP1400-500H	Used for growth medium: 10% FBS, 1% P/S in DMEM
Penicillin/Sreptomycin	VWR	12001-350	Used for growth medium: 10% FBS, 1% P/S in DMEM
Chicken serum	Sigma	C5405	5% chicken serum in CIM is used for blocking solution during immunostaining
PBS (Ca2+ and Mg2+ free)	VWR	12001-664
Epifluorescence microscope	Olympus America, PA	IX81
TIRF illuminator	Olympus America, PA	Cell^TIRF (IEC60825-1:2007)
Epifluorescence illuminator	Sutter Instruments, Novato CA	Lambda DG-4 (DG-4 30)
CCD Camera	Photometrics, Tucson, AZ	Evolve 512 EMCCD
Image acquisition and anaysis software	Intelligent Imaging Innovations, Inc. Denver, CO	Slidebook 5
Pulsed laser	Intelligent Imaging Innovations, Inc. Denver, CO	Ablate (3iL13)
Stage top incubator	Tokaihit, Japan	INUBG2ASFP-ZILCS