Demonstrating Fluorescent Peptidyl Inhibitor Targeting of Mitochondrial ClpXP by Fluorescence Microscopy in Mammalian Cells

Summary

This protocol establishes a quantitative imaging workflow that features the application of confocal microscopy to monitor the selective localization of the fluorescent peptidyl ClpP inhibitor FAM-FAPAL-CMK (Carboxyfluorescein-FAPAL-chloromethyl ketone) into the mitochondria to target ClpP, and the subsequent morphological changes in mitochondria in intact cells caused by ClpP inhibition.

Abstract

Mitochondrial ATP-dependent proteases are essential for maintaining protein homeostasis through degradation of damaged or misfolded proteins. Among these, the ClpXP protease complex locates in mitochondrial matrix and contributes to mitochondrial quality control under physiological and stress conditions. This work demonstrates a quantitative fluorescence microscopy workflow to assess mitochondrial targeting of the fluorescent peptidyl inhibitor FAM-FAPAL-CMK and evaluate mitochondrial morphological changes associated with ClpXP inhibition in mammalian cells. HeLa cells were treated with FAM-FAPAL-CMK and analyzed using confocal microscopy combined with immunofluorescence staining of mitochondrial markers and quantitative image analysis. Colocalization analysis using Costes thresholding and Manders’ overlap coefficients demonstrated mitochondrial enrichments of the inhibitor signal. As a consequence, inhibition of ClpP altered mitochondrial morphology. Immunoblot analysis showed no significant change in ClpP protein abundance upon inhibitor treatment. Taken together, this work describes a reproducible imaging-based workflow that will enable interrogation of mitochondrial ClpXP functions in intact cells in response to perturbations of homeostasis, such as oxidative stress.

Introduction

Mitochondrial ATP-dependent proteases are central to protein homeostasis, degrading misfolded or damaged proteins^1,2,3,4,5,6. Among these, ClpXP is a hetero-oligomeric AAA+ protease complex located in the mitochondrial matrix. The ClpX ATPase recognizes and unfolds proteins, then translocates them to the ClpP proteolytic chamber^7,8,9. Along with other mitochondrial proteases, ClpX....

Protocol

Experiments were performed using commercially available HeLa cells. No human participants, vertebrate animals, or primary human- or vertebrate-derived biological materials were used. The reagents and the equipment used are listed in the Table of Materials.

1. Cell culture and treatment

1. Culture HeLa cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 ˚C in a humidified incubator with 5% CO₂.
2. Plate cells onto sterile glass coverslips and incubate for 24 h to allow cell attachment.
3. Dissolve the inhibitor FAM....

Results

ClpP inhibition alters mitochondrial morphology and network organization
Mitochondrial morphology was assessed to determine whether ClpP inhibition alters mitochondrial organization in HeLa cells. Representative fluorescence images showed clear differences in mitochondrial organization between control and FAM-FAPAL-CMK-treated cells (Figure 1A). Quantitative analysis showed that mitochondrial area decreased from 1.15 ± 4.8 µm² (n = 8013) in control cells to 0........

Discussion

The peptidyl chloromethyl ketone FAPAL-CMK¹⁵ has previously been shown to be a potential irreversible inhibitor of the protease subunit (ClpP) of the mitochondrial ATP-dependent protease ClpXP. While the kinetic experiments demonstrated the inhibitor’s potency and the mechanism of action, the in vitro approach could not evaluate the efficacy of FAPAL-CMK as a selective mitochondrial ClpP inhibitor in intact cells. Fluorescence confocal microscopy confirmed that FAM-FAPAL-CMK was sel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 647-conjugated rabbit secondary antibody	Jackson Immuno Research	711-607-003
Beta Actin mouse antibody	Proteintech	66009
bovine serum albumin	Fisher bioreagents	BP1600
CHAPS	VWR	M127
ClpP rabbit PolyAb	Proteintech	15698
cOmplete protease inhibitor cocktail	Roche	11697498001
D-PBS	Corning	21-031-CV
Dulbecco’s Modified Eagle Medium	Corning	10-013-CV
Dylight405-conjugated mouse secondary antibody	Jackson Immuno Research	715-475-150
ECL Select Western Blotting Detection Reagent	Cytiva	RPN2235
EGTA	Calbiochem	4100
fetal bovine serum	R&D Systems	S11550
glutaraldehyde, 70% solution	Polysciences, Inc	01201-2
GraphPad Prism	11.0.2 (100)
HEPES	Dot Scientific, Inc	DSH75030
HRP-conjugated nouse antibody	Cell Signaling Technology	7076S
HRP-conjugated rabbit antibody	Cell Signaling Technology	7074S
ImageJ/Fiji	2.16.0/1.54p
Live cell imaging solution	Gibco	A59688DJ
Magnesium Chloride Hexahydrate	Fisher	BP214
Mitochondria-Analyzer plugin for Fiji	https://github.com/AhsenChaudhry/Mitochondria-Analyzer
MitoTracker DeepRed	Life Technologies	M22426
mouse anti-TOMM20	Santa Cruz Biotechnology	SC17764
paraformaldehyde, 16% solution	Electron Microscopy Sciences	15710
PIPES	Dot Scientific, Inc	DSP40140
PMSF	RPI research products	P20270
Potassium Chloride	Alfa Aesar	11595
potassium phosphate monobasic	VWR	BDH9268
ProLong mounting medium	Invitrogen	P36930
Protein Assay Dye	Bio-Rad	5000006
saponin	Thermo Scientific	A18820.14
sodium borohydride	Thermo Scientific	200050250
Sodium Chorlde	VWR	BDH9286
sodium deoxycholate	Sigma	D6750
Sodium Dodecyl Sulfate	VWR	0227
sodium phosphate dibasic	AMERSCO	0348
Software name	Version information
Tris	Dot Scientific, Inc	DST60040
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)	GOLD BIOTECHNOLOGY INC	TCEP-10
Triton X-100	Sigma	X100
Tween 20	VWR	0777
ZEISS Zen	2.3