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, ClpXP maintains protein quality control under both normal and stress conditions, including oxidative stress and metabolic perturbations^1,2,4. Disruption of mitochondrial protein homeostasis has been linked to diseases such as neurodegeneration and cancer, underscoring the importance of understanding these protease activities in intact cellular systems^3,6,10.

A major challenge in the field is the lack of methods to directly monitor mitochondrial protease activity with spatial resolution in intact cells. Existing approaches rely primarily on biochemical assays using purified proteins or cell lysates, which provide detailed kinetic information but do not preserve subcellular context^11,12,13,14. Chemical biology strategies have addressed this limitation in part by developing fluorogenic peptide substrates and active-site-directed inhibitors. Fluorescent peptidyl substrates enable quantitative measurement of ATP-dependent peptidase activity, while peptidyl inhibitors form reversible covalent interactions with the proteolytic site to achieve selective inhibition^{13,15,16,17,18,19}. Previously, the lab-synthesized ClpXP inhibitor FAPAL-CMK has been shown to form a covalent bond with ClpP²⁰. In this study, FAM-FAPAL-CMK, a fluorescein-labeled derivative of FAPAL-CMK, was developed as a mechanism-based inhibitor of ClpP, enabling visualization of its intracellular localization.

Compared with biochemical assays, confocal fluorescence microscopy enables spatially resolved assessment of probe distribution and mitochondrial morphology in intact cells, providing information that cannot be obtained from lysates alone. However, unlike enzymatic assays, fluorescence localization does not directly measure catalytic activity or molecular binding events and may be influenced by probe uptake efficiency, mitochondrial dynamics, and imaging conditions. In addition, successful implementation depends on adequate probe accumulation and sufficiently resolved mitochondrial networks, making this workflow most suitable for adherent cultured cells with clearly distinguishable mitochondrial morphology.

The goal of this study is to establish a quantitative workflow that integrates mechanism-based fluorescent probes with confocal microscopy to monitor mitochondrial protease localization and function in intact cells. By combining selective chemical probes with high-resolution confocal imaging, this workflow enables assessment of mitochondrial localization, probe distribution, and morphological responses to perturbations of mitochondrial homeostasis in intact cells. It can complement the existing biochemical assays that directly measure protease activity but lack spatial resolution.

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-FAPAL-CMK in DMSO to prepare a 50 µM stock and freeze at -20 °C for storage.
4. Add 50 nM FAM-FAPAL-CMK to the cell growth medium in the treated cell samples.
5. Add an equal volume of DMSO to control samples to match the final solvent concentration of inhibitor-treated samples, ensuring that any effects are not due to DMSO.
6. Incubate the cells for an additional 18 h under the same culture conditions prior to downstream processing.
   Note: FAM-FAPAL-CMK inhibitor is lab-designed and synthesized, and the synthesis method has been reported previously²⁰.

2. Fixation and permeabilization

1. Wash the cells with pre-warmed D-PBS for 5 min and repeat once.
2. Fix the cells for 15 min at room temperature using PHEM buffer (68 mM PIPES, 25 mM HEPES, 15 mM EGTA, 3 mM MgCl₂, pH 6.9) containing 2.4% (v/v) formaldehyde, 0.05% (v/v) glutaraldehyde, and 0.5% (w/v) saponin.
3. Prepare fresh sodium borohydride solution (5 mg/mL in D-PBS).
4. Incubate the cells in the sodium borohydride solution for 15 min at room temperature to neutralize residual glutaraldehyde.
5. Rinse cells three times with D-PBS for 5 min each prior to staining.
   NOTE: Formaldehyde and glutaraldehyde are toxic fixatives. Perform fixation steps in the hood and wear appropriate personal protective equipment (gloves, lab coat, and eye protection). Sodium borohydride is a reactive reducing agent and should be handled carefully. The PHEM-based fixation buffer containing formaldehyde, glutaraldehyde, and saponin was selected to preserve mitochondrial morphology while maintaining antigen accessibility during immunostaining. Formaldehyde and glutaraldehyde provide protein crosslinking for structural preservation, while the buffering composition and pH of PHEM help stabilize mitochondrial ultrastructure²¹. Saponin facilitates membrane permeabilization to improve the rate of fixative penetration. This fixation strategy minimizes distortion of mitochondrial networks and is particularly suitable for quantitative analysis of mitochondrial morphology.

3. Immunofluorescence staining

1. Block cells for 1 h at room temperature in blocking buffer containing 3% (w/v) bovine serum albumin (BSA) in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) with 0.1% (v/v) Triton X-100.
2. Incubate cells with primary antibodies diluted in blocking buffer for 1 h at room temperature. Use mouse anti-TOMM20 (1:500) and rabbit anti-ClpP (1:500).
3. Wash cells three times with PBS for 5 min each to remove unbound primary antibodies.
4. Incubate cells with secondary antibodies diluted in blocking buffer for 1 h at room temperature. Use CF405S-conjugated mouse secondary antibody (1:500) and Alexa Fluor 647-conjugated rabbit secondary antibody (1:500).
5. Protect samples from light during and after secondary antibody incubation.
6. Repeat step 3.3.
7. Mount coverslips onto glass slides using mounting medium.
   NOTE: ClpP and TOMM20 were visualized under 405 nm and 647 nm to avoid any spectral overlap with FAM signals.

4. Image acquisition

1. Acquire images using a confocal laser scanning microscope equipped with a high-numerical-aperture oil-immersion objective.
2. Excite fluorophores using 405 nm and 633 nm laser lines. Use appropriate dichroic beam splitters to reflect excitation light and transmit emitted fluorescence to the detectors.
3. Set the pixel size to 35.3 nm (X, Y) and the Z-step to 0.1 µm to ensure sufficient axial sampling of mitochondrial structures.
4. Acquire images with a frame size of 1900 x 1900 pixels (X, Y) and collect 5 optical sections per Z-stack beginning at the upper mitochondrial signal and ending below the mitochondrial signal to ensure the predominant mitochondrial signal is collected.
   NOTE: Representative microscope acquisition settings are provided in Supplementary Figure 1. For HeLa cells, mitochondria are typically thin tubular structures, around 0.5-1 µm in diameter²². So a 0.5 µm Z-volume is enough to capture a substantial fraction of the individual mitochondrial matrix, but not necessarily the entire cell. A 5-section Z-stack was selected to balance sufficient sampling of mitochondrial structures with rapid image acquisition to minimize motion-related artifacts during cell imaging.

5. Live cell treatment and imaging

1. Culture HeLa cells on 35 mm glass-bottom dishes in DMEM with 10% FBS at 37 °C in a humidified incubator with 5% CO₂.
2. Add inhibitor (final concentration: 50 nM) or an equivalent volume of DMSO to cells 24 h after seeding. Incubate cells for an additional 18 h under standard culture conditions.
3. Remove culture medium containing the inhibitor or DMSO and rinse the cells once with pre-warmed D-PBS for 5 min.
4. Add live-cell imaging solution containing 20 nM MitoTracker DeepRed.
5. Perform live-cell imaging at room temperature using the same microscope settings described above. Acquire images in per-frame scanning mode with an approximate 4.72 s per optical section (about 24 s total per 5-section Z-stack).
   NOTE: Five optical sections per Z-stack are set to balance sufficient sampling of mitochondrial structures with manageable acquisition times. Additionally, a room-temperature live-cell imaging solution is used to reduce mitochondrial motility compared to imaging at 37 °C. Although mitochondrial movement during live-cell imaging may still contribute to variability in colocalization measurements, consistent acquisition settings across all samples help to reduce systematic bias. Representative software settings are shown in Supplementary Figure 2.

6. Mitochondrial morphology analysis

1. Open image files in Fiji²³ and generate maximum intensity projections from Z-stacks by selecting Image → Stacks → Z project and choosing Max Intensity.
2. Apply the Otsu thresholding method by navigating to Image → Adjust → Threshold and selecting Otsu from the thresholding options. Apply the threshold to segment mitochondria.
3. Run the Mitochondria Analyzer plugin²⁴ and select 2D analysis mode on a per-mito basis.
4. Measure mitochondrial size (area) and perimeter using the Mitochondria Analyzer output.
5. Obtain the mitochondrial form factor values directly from the Mitochondria Analyzer output, where form factor is calculated as P²/(4πA), with P representing the perimeter and A the area.
6. Measure total branch length per mitochondrion using the network analysis function within the plugin to assess mitochondrial connectivity.
7. Present the data as distributions of individual mitochondrial measurements along with mean values derived from 30 cells per condition across three independent biological replicates.
8. Evaluate the statistical significance using Welch’s t-test in a statistical analysis software.

7. Colocalization analysis

1. Open image files in Fiji and generate maximum intensity 2D projections from 3D image stacks. For consistency, analyze all samples using the same projection and acquisition strategy.
2. Split fluorescence channels by selecting Image → Color → Split Channels.
3. Launch the Coloc 2 plugin by selecting Plugins → Colocalization → Coloc 2.
4. Assign the appropriate channels for analysis and enable Costes threshold regression in the plugin settings. Run the analysis on a per-cell basis.
5. Record Manders’ overlap coefficients (M1 and M2) to assess the fraction of signal in each channel that spatially coincides with the other.
6. Enable Costes randomization in the Coloc 2 settings and set the number of randomizations to 50 to assess statistical significance.

8. Cell lysate generation

1. Seed HeLa cells in DMEM with 10% FBS and incubate at 37 °C in a humidified incubator with 5% CO₂.
2. Add 50 nM inhibitor or equivalent volume of DMSO to the cells 24 h after seeding, and incubate for an additional 18 h under standard culture conditions.
3. Remove the culture medium and rinse cells with pre-warmed D-PBS for 5 min, and trypsinize to detach.
4. Quench trypsinization with fresh cell growth medium and centrifuge at 300 x g for 3 min at room temperature. Discard the supernatant.
5. Resuspend the cell pellet in D-PBS and centrifuge at 300 x g for 3 min to wash cells. Discard the supernatant.
6. Resuspend the cell pellet in pre-chilled lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM sodium chloride, 0.1% SDS, 0.5% sodium deoxycholate, 2% CHAPS, 10 mM TCEP, 1 mM PMSF, 1x protease inhibitor cocktail).
7. Incubate lysates on ice for 20 min and sonicate lysates in an ice-water bath for 2 min to ensure complete lysis.
8. Centrifuge lysates at 13,000 x g, 4 °C for 20 min.
9. Transfer the cleared supernatant to a fresh pre-chilled tube for downstream analysis.
   NOTE: PMSF is toxic and should be handled in a fume hood with appropriate personal protective equipment. SDS is an irritant, and sonication generates heat and aerosols. Perform lysis and sonication steps on ice and follow institutional safety guidelines.

9. Bradford assay

1. Prepare a set of standard solutions using BSA in 0.15 M sodium chloride. The concentrations are: 0, 0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, 0.10 mg/mL.
2. Dilute the unknown cell lysate 50-fold and 100-fold in 0.15 M sodium chloride.
3. Pipette replicates of each standard curve solution and unknown samples into a 96-well plate.
4. Add Bradford reagent to each well. For each well, use 160 µL protein solution with 40 µL 5x Bradford reagent (commercially available). Mix gently by pipetting.
5. Incubate the plate at room temperature for 10 min and measure absorbance at 595 nm using a plate reader.
6. Generate a standard curve by plotting BSA concentrations on the x-axis and the averaged absorbance at 595 nm from replicate measurements on the y-axis.
7. Determine protein concentrations of samples using the standard curve. Multiply calculated values by the dilution factor to get the original concentration.

10. Immunoblotting

1. Load equal amounts of cell lysate onto a 12% polyacrylamide gel and separate proteins by SDS-PAGE.
2. Activate the PVDF membrane by briefly immersing it in ethanol. Equilibrate both the membrane and blotting papers in the transfer buffer.
3. Transfer proteins from the gel to a PVDF membrane using a semi-dry transfer system.
4. Block the membrane in 5% (w/v) non-fat dry milk in TBS (20 mM Tris, 150 mM NaCl, pH 7.6) at room temperature for 1 h with gentle agitation.
5. Incubate the membrane with the primary antibody diluted in blocking buffer at room temperature for 1 h with gentle agitation. Use rabbit anti-ClpP (1:2000) and mouse anti-beta-actin (ACTB, 1:10,000).
6. Wash the membrane three times with TBST (20 mM Tris, 150 mM NaCl, 0.1% (v/v) Tween 20, pH 7.6) for 5 min per wash to remove unbound antibody.
7. Incubate the membrane with HRP-conjugated secondary antibody diluted in blocking buffer (1:3000) at room temperature for 1 h with gentle agitation.
8. Repeat the wash step.
9. Detect protein bands using chemiluminescent detection reagents.

11. Densitometric analysis

1. Open the Western blot image in Fiji.
2. Change the image type to 8-bit by Image → type → 8-bit.
3. Subtract the image background by Process → subtract background. Check light background and set the rolling ball radius to 50.0 pixels.
4. Circle out the band at the first lane using the rectangle tool, then select analyze → gel → select first lane.
5. Move the rectangle tool to the second lane, and select analyze → gel → select next lane. Repeat this step until all lanes are selected.
6. Plot the band density by analyze → gel → plot lanes.
7. Use the straight line tool to draw a line at the bottom of the band density peak, and use the wand tool to read the band density.
8. Repeat the same procedure from steps 11.1–11.8 on the loading control blot image.
9. Report the relative band density by dividing density (ClpP) by density (ACTB).

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