Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 derived vesicles and mitochondria to the injured cell membrane^1-7. However, the details of the sequence of molecular and subcellular events involved in repairing damaged cell membrane remains poorly understood.

Cell's repair response can be segregated into early and late responses. Early responses, which occur within seconds to minutes time scale, are tremendously important in determining the nature of late responses leading to successful cell repair or cell death. End point assays based on bulk biochemical and cellular analysis have helped establish the involvement of molecular and cellular processes in repair. But, due to the heterogeneity and rapidity of cellular repair response, end point assays fail to provide the kinetic and spatial details of the sequence of events leading to repair. Approaches that enable controlled injury of cell membrane and allow monitoring the cell membrane repair and associated subcellular responses at high spatial and temporal resolution are ideally suited for such studies. Here, such approaches have been presented. Two of the protocols describe approaches to monitor the real time kinetics of cell membrane repair and the subcellular responses associated with the repair process in live cells following laser injury. As a complement to these live cell imaging based assays, end point assays have also been described that provide a population based measure for monitoring repair of individual cells and the associated subcellular responses. To demonstrate their utility these approaches have been used to monitor trafficking and exocytosis of lysosomes in response to cell membrane injury.

Protokół

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 transfer coverslips on silicone O-rings.
2. Add 100 μl of prewarmed FITC dextran solution in CIM on C1 and C2 or in PBS on C3.
3. Injure plasma membrane on C1 and C3 by gently rolling 40 mg glass beads over the coverslip at room temperature by manually tilting coverslips back and forth 6-8 times at an angle of 30°.
   Note: For mild injury, ensure the glass beads are spread uniformly, thus minimizing repeat injuries. To improve reproducibility of injury between samples, simultaneously carry out the glass bead injury of the different samples to be compared.
4. Avoiding further rolling of beads, transfer all coverslips to a 37 °C incubator at ambient CO₂ and allow repair to proceed for 5 min.
5. Without letting the beads roll, remove the glass beads and FITC dextran by washing with CIM (C1 and C2) or PBS (C3) at 37 °C.
6. Place coverslips back on the O ring and add 100 μl of prewarmed lysine fixable TRITC dextran solution in CIM on C1 and C2 or in PBS on C3.
7. Incubate at 37 °C for 5 min at ambient CO₂.
8. Wash coverslips twice with prewarmed CIM and fix with 4% PFA for 10 min at RT.
9. Wash twice with PBS and incubate for 2 min at RT in Hoechst dye.
10. Wash samples twice with PBS, mount on a slide using mounting media and image cells using an epifluorescence microscope.
11. Use cells from C2 to determine the background red and green staining intensity and use this value to threshold the red and green channels for all samples (C1-C3).
12. Score total number of cells that are a) green (injured and repaired) and b) red or both red and green (injured, but failed to repair) in C1 and C3 samples.
13. Count >100 greens cells for each condition and express the fraction of cells that failed to repair as a percent of all cells injured (green and red).

2. Live Imaging of the Kinetics of Cell Membrane Repair Following Laser Injury

1. Wash cells with prewarmed CIM and then put the coverslip in CIM with FM dye.
2. Place the coverslip in a holder in the stage top incubator maintained at 37 °C.
3. Select a 1-2 mm² region of the cell membrane, and irradiate for <10 msec with the pulsed laser. Attenuate the laser power through the software to 40-50% of the peak power. Optimal power allows consistent, but nonlethal injury and this must be determined by trial and error for each individual instrument and cell line being used.
4. To monitor repair, image every 10 sec in epifluorescence and brightfield, starting prior to injury and continue for 3-5 min  following injury.
   Note: For no repair control, repeat steps 2.1-2.4 with the cells injured in PBS containing FM dye.
5. To quantify the kinetics of repair, measure cellular FM dye fluorescence and plot the change in intensity (ΔF/F0) during the course of imaging. This data should be averaged for over 10 cells in each condition and plotted as averaged or individual cell's value, as needed.

3. Imaging Bulk (Glass Bead) Injury Induced Lysosomal Exocytosis

The samples include following cells grown to>50% confluence: 1. Test (C1) - Cells allowed to repair in presence of Ca²⁺, 2. Control 1 (C2; No injury) - Cells neither injured nor incubated with primary antibody, and 3. Control 2 (C3; No repair) - Cells allowed to repair in absence of Ca²⁺.

1. Wash coverslips C1 and C2 twice with CIM at 37 °C and C3 with PBS at 37 °C and transfer them on silicone O-rings.
2. Add 100 μl of prewarmed lysine fixable TRITC dextran in CIM on C1 and C2 and in PBS on C3.
3. Injure plasma membrane on C1 and C3 as in step 1.3.
4. Avoiding further rolling of beads, transfer all coverslips to a 37 °C incubator at ambient CO₂ and allow repair to proceed for 5 min.
5. Remove the glass beads and TRITC dextran by washing the coverslips in cold growth medium, again ensuring no rolling of the glass beads on the cells.
6. Rinse the coverslips twice with cold growth medium and transfer to the O-rings.
7. To C1 and C3, add 100 μl of rat anti mouse LAMP1 antibody in cold complete growth media and add the cold, complete growth media to C2.
8. To allow antibody binding incubate coverslips for 30 min at 4 °C.
9. Wash the coverslips three times with cold CIM and fix all coverslips with 4% PFA for 10 min  at RT and then rinse 3x with CIM.
10. Incubate all coverslips in 100 μl of blocking solution for 15 min at RT
11. Incubate all coverslips in 100 μl Alexa Fluor 488 anti rat antibody for 15 min at 4 °C.
12. Wash twice with PBS and incubate for 2 min at RT in Hoechst.
13. Wash cells twice with PBS, mount on a slide using mounting media and image using an epifluorescence microscope.
14. Using images of C2 cells, determine the nonspecific background staining in the red (TRITC dextran) and green (Alexa Fluor 488 antibody) channels and use these background staining values to threshold the red and green channels for all samples (C1-C3).
15. Use the >100 red labeled cells from C1 and C3 to measure the intensity of LAMP1 staining (green) in injured cells. For a successful experiment the LAMP1 staining in cells from C3 will be significantly lower than in cells from C1.

4. Live Imaging of Cell Membrane Injury Triggered Subcellular Trafficking

1. Fluorescently label the compartment of interest by transfecting appropriate reporter (e.g. CD63-GFP for lysosomes⁸) or by using fluorescent dyes⁹.
2. For labeling lysosomes with fluorescent dye, incubate cells grown to 50% confluence in growth media containing FITC-dextran
3. Allow dextran to be endocytosed for 2 hr (or longer as needed for sufficient endosomal labeling with dextran by the cell line of interest) in the CO₂ incubator.
4. Wash cells with prewarmed growth media and incubate in it for 2 hr in a CO₂ incubator to allow all endocytosed dextran to accumulate in the lysosome.
5. Before imaging, rinse the coverslip in prewarmed CIM and mount in a coverslip holder on the stage top incubator at 37 °C.
6. Carry out widefield (for movement throughout the cell) or TIRF (movement at the cell surface and exocytosis) imaging - cells that do not have crowded FITC dextran labeled lysosomes are ideal for imaging the movement and exocytosis of individual lysosomes.
7. Aligning the TIRF lasers for imaging microscope: Use 60X or 100X objective with >1.45 NA. Set up the angle for incident TIRF laser beam using the manufacturer's approach.
8. Injure the cell membrane by irradiating a small (1-2 mm²) region for <100 msec, with the pulsed laser at 40-50% attenuation.
9. To monitor the response of lysosome to cell injury, image cells at 4-6 frames/sec for at least 2 min or longer depending on the dynamics of exocytosis in the cells of interest.

Wyniki

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 ...

Dyskusje

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...

Materiały

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