Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 11, pp. 2028-2036 The Author(s) 2024. This article is an open access publication.

2028

Vimentin and Desmin Intermediate Filaments

Maintain Mitochondrial Membrane Potential

Alexander A. Dayal

, Olga I. Parfenteva

, Wang Huiying

, Anton S. Shakhov

1,2

Irina B. Alieva

1,2

, and Alexander A. Minin

1,a

Institute of Protein Research, Russian Academy of Sciences, 119334, Moscow, Russia

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

e-mail: alexminin@gmail.com

Received August 28, 2024

Revised October 4, 2024

Accepted October 9, 2024

Abstract— Intermediate filaments (IFs) represented by a diverse range of proteins, are one of the three main

cytoskeleton components in different types of animal cells. IFs provide mechanical strength to cells and help

position the nucleus and organelles in the cell. Desmin is an IF protein typical of muscle cells, while vimentin,

which has a similar structure, is expressed in many mesenchymal cells. Both proteins are synthesized during

myogenesis and regeneration of damaged muscle tissue and form a mixed IF network. Both desmin and vi-

mentin regulate mitochondrial activity, including mitochondrial localization and maintenance of mitochondrial

membrane potential, in the corresponding cells, but the role of mixed IFs in the control of mitochondrial

functions remains unclear. To investigate how a simultaneous presence of these proteins affects mitochondrial

membrane potential, we used BHK21 cells expressing both vimentin and desmin IFs. Expression of vimentin

or desmin individually or both proteins simultaneously was suppressed using gene knockout and/or RNA

interference. It was found that disruption of biosynthesis of either vimentin or desmin did not affect the

mitochondrial membrane potential, which remained unchanged compared to cells expressing both proteins.

Simultaneous abolishment of both proteins resulted in a 20% reduction in the mitochondrial membrane po-

tential, indicating that both vimentin and desmin play an equally important role in its maintenance.

DOI: 10.1134/S0006297924110154

Keywords: mitochondria, vimentin, desmin, mitochondrial membrane potential

Abbreviations: IF, intermediate filament; MMP, mitochon-

drial membrane potential.

* To whom correspondence should be addressed.

INTRODUCTION

Mitochondria are eukaryotic cell organelles that

play a critical role in cell functioning under both nor-

mal and pathological conditions. They are essential

for ATP synthesis, generation of reactive oxygen spe-

cies, regulation of apoptosis and intracellular calcium

concentration, and numerous other cellular functions

[1-4]. At the organismal level, mitochondria are in-

volved in the immune response, stress response, and

adaptation to exercise, as well as play an important

role in the development of various pathologies, includ-

ing cancer and aging  [5-10]. Most mitochondrial func-

tions directly depend on the mitochondrial membrane

potential (MMP), which is maintained by the respirato-

ry chain complexes located in the inner mitochondrial

membrane  [11]. Electron transport and proton trans-

fer across the membrane during respiration result in

the establishment of the electrical potential of about

–180  mV between the cytosol and the mitochondrial

matrix  [11]. The importance of maintaining the MMP

is emphasized by the fact that depolarized mitochon-

dria, i.e., those with unacceptably low membrane

potential, are selectively eliminated through mitopha-

gy  [12]. Extensive depolarization of mitochondria can

activate apoptosis, resulting in cell death  [13].

Numerous intracellular factors involved in the

MMP regulation have been identified  [14], including

intermediate filament(IF) proteins that play a signifi-

cant role in this process  [15]. For example, mutations

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BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

in keratins have been shown to cause mitochondri-

al fragmentation in hepatocytes, leading to the ag-

gregation of mitochondria and impaired respiratory

function [16-18]. Vimentin (type III IF protein) regu-

lates MMP in fibroblasts; its removal decreases MMP

by 20% [19]. Desmin, an IF protein specific for mus-

cle cells, has also been demonstrated to participate

in the mitochondrial respiratory function  [20-24].

Previously, we found that recombinant vimentin and

desmin expressed and isolated from bacteria, bound

to mitochondria in  vitro  [25,  26], suggesting that they

may be directly involved in regulating mitochondrial

properties.

Although vimentin is typically found in various

mesenchymal cells, while desmin IFs are characteris-

tic of muscle cells, these proteins can form mixed IF

networks under certain conditions. For instance, vi-

mentin and desmin are expressed simultaneously at

the early stages of muscle fiber differentiation and

during regeneration  [27]. Therefore, both vimentin

and desmin can be present in the same cell, as well

as interact with mitochondria and influence their

properties.

The role of vimentin in regulating MMP in the

presence of desmin has been investigated insufficient-

ly. Although several studies have provided compelling

evidence that desmin is involved in the distribution,

morphology, and respiratory function of mitochon-

dria [20-23], its role in the MMP regulation remains

poorly understood. Here, we studied the role of vi-

mentin and desmin in this process in BHK21 cells

expressing both proteins. By selectively suppressing

expression of either desmin or vimentin using RNA

interference (RNAi) and/or the CRISPR-Cas9 system,

we found that each protein could independently

maintain the MMP.

MATERIALS AND METHODS

Cell culture. BHK21 cells and two derivative

cell lines generated using the CRISPR/Cas9 system,

BHK21(Vim

–/–

) and BHK21(Des

–/–

), were cultured in

DMEM (PanEco, Russia) supplemented with 10% fetal

bovine serum (Biolot, Russia), penicillin (100  µg/ml),

and streptomycin (100  µg/ml) (Sigma-Aldrich, USA) at

37°C in a humidified atmosphere with 5%  CO

. For mi-

croscopy, the cells were seeded on sterile coverslips

and incubated for 16-20 h.

RNAi. To deplete desmin in BHK21 cells via

RNAi, we used the pG-SHIN2-des plasmid encoding

shRNA 5′-AAGCAGGAGAUGAUGGAGU-3′ [28] and GFP

as a reporter. Vimentin was knocked down using

the pG-SHIN2-vim plasmid encoding shRNA 5′-CAGA-

CAGGAUGUUGACAAU-3′ [29,  30] (kindly provided by

Prof.  R.  Goldman; Northwestern University, Chicago).

Control cells were transfected with the pG-SHIN2-scr

plasmid encoding scrambled shRNA sequence of the

same length (5′-AUGUACUGCGCGUGGAGA-3′).

Vimentin and desmin knockouts. To knock out

the vimentin gene, BHK21 cells, were transfected

with the pSpCas9n(BB)-Puro-(1+2)Vim plasmid en-

coding two guide RNAs: 5′-CACCGAACTCGGTGTTGAT-

GGCGT-3′ and 5′-CACCGAACACCCGCACCAACGAGA-3′

[31]. The gene for desmin was knocked out using the

pSpCas9(BB)-Puro-Des plasmid encoding the guide

RNA 5′-CACCGCGGCGACCCGGGUCGGCUCG-3′. Cell were

transfected using Transfectin reagent (Evrogen, Rus-

sia) in complete DMEM medium. Briefly, 1 µg of plas-

mid DNA was mixed with 1  µl of Transfectin in 0.1  ml

of serum-free DMEM and added to cells in 1  ml of

complete DMEM. BHK21(Vim–/–) and BHK21(Des–/–)

cells were selected in DMEM containing 2  µg/ml pu-

romycin and 1 µg/ml verapamil.

Fluorescent microscopy of live cells. Mito-

chondria were stained by incubating cells with 5  nM

tetramethylrhodamine (TMRM; Molecular Probes,

USA) in the presence of 2.2  µM verapamil for 30  min

at 37°C. Following incubation, the coverslips with

the cells were placed in a sealed chamber contain-

ing DMEM and imaged with a Keyence BZ-9000 mi-

croscope (USA) equipped with an incubator for live-

cell imaging. The temperature in the incubator was

maintained at 36  ±  2°C. The cells were imaged with a

PlanApo 63× objective and a 12-bit digital CCD camera.

The images were transferred to a computer using the

BZ II Viewer software (Keyence, USA) and saved as

12-bit graphic files for further analysis.

Immunofluorescence and immunoblotting. For

IF staining, the cells were fixed with methanol at –20°C

for 10min and incubated with mouse monoclonal an-

ti-vimentin antibodies V9 (Sigma-Aldrich) and mouse

monoclonal anti-desmin antibodies DE-U-10 Sigma-

Aldrich). FITC- and TRITC-conjugated anti-mouse sec-

ondary antibodies (Jackson, USA) were used for pro-

tein detection. Microphotographs were acquired using

a Keyence BZ-9000 microscope (USA) with a PlanApo

63× objective and a 12-bit digital CCD camera.

Super-resolution structured illumination micros-

copy (SR-SIM) was performed with a Nikon N-SIM mi-

croscope (Nikon, Japan) with a ×100/1.49  NA oil im-

mersion objective and a 561  nm diode laser; Z-stacks

were acquired at 0.12-µm intervals with an EMCCD

camera (iXon 897, Andor, Japan). Exposure was opti-

mized to maintain an average brightness of ~5000  a.u.

to minimize photobleaching. Images were acquired

with the NIS-Elements 5.1 software (Nikon).

SDS-PAGE was conducted according to Laemmli’s

method [32], followed by immunoblotting as previous-

ly described  [26]. The membranes were stained with

V9 antibodies (vimentin), DE-U-10 antibodies (desmin),

and DM1A monoclonal antibodies (tubulin) and then

DAYAL et al.2030

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig.  1. SR-SIM imaging of vimentin (green) and desmin (red) IFs in BHK21 cells (immunostaining). Scale bar: 10 µm.

with secondary anti-mouse antibodies conjugated to

horseradish peroxidase (Jackson). Protein bands were

visualized using hydrogen peroxide and diaminoben-

zidine as peroxidase substrates.

MMP measurements. MMP was evaluated by

measuring TMRM fluorescence as reported in [19]. Mi-

tochondrial contours were defined using the “analyze

particles” plugin of the ImageJ software in a region

with a single layer of cells, and the average fluores-

cence intensity of all pixels within each contour was

calculated. For each experiment, 10-15 regions each

containing 15-40 mitochondria were analyzed. The

data are presented as the mean fluorescence intensity

for all mitochondria ± standard error. The normality

of data distribution was assessed with the Shapiro–

Wilk test; the homogeneity of variance was checked

with the F-test. The significance of differences was

estimated with the paired Student’s t-test.

RESULTS

Fibroblast-like BHK21 cells contain IFs com-

posed of vimentin and desmin (type III proteins),

with small amounts of nestin (type VI protein)  [33].

As demonstrated by the high-resolution immunofluo-

rescence microscopy, these filaments were formed by

co-polymerization of vimentin and desmin. Figure  1

shows that the distribution of these proteins was not

perfectly uniform, suggesting different proportions

of vimentin and desmin in individual filaments. IFs

in the perinuclear region were enriched in desmin,

while vimentin predominantly localized to the cell pe-

riphery. The overall organization of IFs was a radial

network, which implies participation of the microtu-

bule transport system in the intracellular distribution

ofIFs. To investigate the role of desmin and vimentin

in regulating mitochondrial function, we decided to

selectively delete each protein from the cells.

First, we assessed how disruption of vimentin or

desmin biosynthesis would affect the IF network in

BHK21 cells. Using RNAi with the plasmids based on

the pG-SHIN2 carrying the GFP reporter gene [29,  30],

we generated BHK21 cells with IFs composed solely

of vimentin or desmin. As shown in Fig.  2, expres-

sion of the corresponding interfering RNAs resulted in

almost complete absence of desmin (Fig.  2, a and b)

or vimentin (Fig.  2, c and d) in the transfected cells

(identified by co-expression of GFP). In contrast, con-

trol cells transfected with pG-SHIN2-scr displayed no

changes in the levels of either desmin (Fig.2,e andf)

or vimentin (not shown).

Previously, we found that suppression of vimen-

tin biosynthesis in fibroblasts led to a decrease in the

MMP  [16]. Here, we investigated the effect of vimentin

knockdown in BHK21 cells also expressing desmin and

found that the absence of vimentin did not affect the

MMP in these cells. (Fig.  3). This suggests that desmin

was able to maintain the MMP at a high level, similar

to vimentin. The knockdown of desmin did not lead

to the decrease in the MMP either (Fig.  3), likely due

to the presence of vimentin in the cells. These data

suggest that both vimentin and desmin independently

regulate MMP in BHK21 cells.

The next step in testing our hypothesis was to as-

sess the MMP in cells lacking both proteins. To achieve

this, we used a two-step approach. First, BHK21 cells

expressing vimentin or desmin only were generated

using the CRISPR-Cas9 system. Next, the remaining

protein was knocked down by RNAi. The data of

Western blotting (Fig.  4) showed that the obtained

BHK21(Des

–/–

) and BHK21(Vim

–/–

) cell lines completely

lacked the knocked-out proteins and, according to im-

munofluorescence microscopy analysis, contained IFs

composed solely of vimentin (Fig. 5, a, b) or desmin

(Fig.5,c,d), respectively. Vimentin IFs in BHK21(Des

–/–

)

cells form a normal, uniformly distributed radial net-

work. In contrast, desmin IFs in BHK21(Vim

–/–

) cells

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BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig.  2. Disruption of desmin (a, b) and vimentin (c, d) IFs by RNAi in BHK21 cells transfected with pG-SHIN2-des and

pG-SHIN2-vim plasmids, respectively. Control cells(e,f) were transfected with pG-SHIN2-scr. IFs were stained with antibodies

against desmin (Des) and vimentin (Vim). Transfected cells were identified by GFP expression. Scale bar: 10 µm.

were partially aggregated, suggesting that the proper

distribution of desmin IFs depends on the presence

of vimentin.

Transfection of BHK21(Des

–/–

) cells with the plas-

mid encoding interfering RNA against vimentin al-

lowed us to significantly reduce the content of this

protein and to obtain the cells lacking vimentin IFs

(Fig.  6, a and b). However, complete removal of de-

smin IFs in BHK21(Vim

–/–

) cells proved to be more

challenging. As shown in Fig.  6, c and d, the trans-

fected cells contained desmin aggregates, while the

filament network at the cell periphery was entirely

absent. In contrast, cells transfected with the control

plasmid retained the IF network (data not shown).

Disruption of vimentin biosynthesis in

BHK21(Des

–/–

) cells decreased the MMP compared

to the control cells (Fig.  7a), indicating involvement

of vimentin IFs in the MMP maintenance. A similar

effect was observed when desmin expression was

suppressed in BHK21(Vim

–/–

) cells, even though de-

smin was not completely eliminated from the cells.

Presumably, the remaining desmin aggregates did

not impact the MMP. Interestingly, the reduction in

the MMP (~20%) was similar in BHK21(Des

–/–

) and

DAYAL et al.2032

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig.  3. RNAi-mediated knockdown of desmin (shRNA-des) and

vimentin (shRNA-vim) in BHK21 cells did not affect the MMP.

Control cells were transfected with pG-SHIN2-scr. The data

are presented as mean fluorescence intensity of mitochon-

dria in the indicated number of cells ± standard error, ex-

pressed as a percentage of the average fluorescence intensity

in non-transfected cells (none); p > 0.10.

Fig.  4. Western blot analysis of cell homogenates from the

original BHK21 cells and BHK21(Des

–/–

) and BHK21(Vim

–/–

)

cell lines generated by the knock-out of desmin and vimen-

tin genes, respectively. Alpha-tubulin was used as a loading

control.

BHK21(Vim

–/–

) cells, suggesting that both IF proteins

independently contribute to the MPP maintenance

in BHK21 cells.

DISCUSSION

Various IF proteins play a critical role in the mi-

tochondrial motility, shape, and functions in different

types of cells [34,  35]. However, the molecular mech-

anisms governing the interaction between IF proteins

and mitochondria remain largely unexplored. Analysis

of amino acid sequences of IF proteins has identified

in some of them regions that could function as mi-

tochondrial localization signals [19,  25,  26]. Hence, at

least some IF proteins can bind directly to the mi-

tochondria, as was demonstrated in in  vitro experi-

ments for vimentin and desmin [25,  26]. In this study,

Fig.  5. IFs in BHK21(Des

–/–

) (a, b) and BHK21(Vim

–/–

) (c, d) cells. Immunofluorescent staining with anti-vimentin and anti-

desmin antibodies. Scale bar: 10 µm.

INTERMEDIATE FILAMENTS MAINTAIN MITOCHONDRIAL MEMBRANE POTENTIAL 2033

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig.  6. RNAi-mediated deletion of vimentin (a,b) in BHK21(Des

–/–

) cells and desmin(c,d) in BHK21(Vim

–/–

) using transfection

with the pG-SHIN2-vim and pG-SHIN2-des plasmids, respectively. IFs were visualized by staining with anti-vimentin (b) and

anti-desmin(d) antibodies. Transfected cells (indicated by arrows) were identified by GFP expression(a,c). Scale bar: 10µm.

Fig. 7. Decrease in the MMP in BHK21(Vim

–/–

) cells(a) and BHK21(Des

–/–

) cells(b) as a result of RNAi. BHK21 cells transfect-

ed with the pG-SHIN2-scr plasmid were used as a control. The data are presented as mean fluorescence intensity of mito-

chondria in the indicated number of cells ± standard error, expressed as a percentage of the average fluorescence intensity

in non-transfected cells; p < 0.05.

we used BHK21 cells expressing both vimentin and

desmin to show that desmin can maintain a high

MMP, similar to vimentin, which had been previously

shown to possess this ability as well [19]. Based on

these data, it can be concluded that mitochondria

in muscle cells can function normally both in the

DAYAL et al.2034

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

presence of desmin IFs only and in the case of simul-

taneous expression of vimentin and desmin. Expres-

sion of vimentin gene in muscle cells seems to have

no effect on the functioning of mitochondria, at least

not on their membrane potential.

It remains unclear how the interaction between

mitochondria and vimentin or desmin IFs leads to

the MMP increase. It can be suggested that despite

a relatively small effect of IFs on the MMP, their in-

fluence on the mitochondrial properties is still sig-

nificant  [35]. The data from this study indicate that

both proteins, whether acting together or individual-

ly, have a similar impact on the membrane potential.

This implies that vimentin and desmin may operate

through a common mechanism, otherwise, an addi-

tive effect would be expected. As mentioned above,

both proteins contain regions that could serve as mi-

tochondrial localization signals [19,  25,  26] potentially

recognized by mitochondrial complexes responsible

for protein import. Future research will determine

whether these complexes are involved in regulating

the MMP.

CONCLUSION

Our data also suggest that vimentin expression

in muscle cells during differentiation or regeneration

after injury may play a role in the establishment of

normal desmin IF network, as follows from the partial

aggregation of desmin IFs in the vimentin-deficient

cells. However, further studies are needed to validate

this hypothesis.

Acknowledgments. The authors express their

gratitude to J. Agnetti (Bologna, Italy) for kindly pro-

viding anti-desmin antibodies, R. Goldman (Chicago,

USA) for providing genetic constructs, I. Kireev (Lo-

monosov Moscow State University, Russia) for help

with the high-resolution microscopy, and N. Minina

for her skilled technical assistance.

Contributions. A.A.M. developed the concept

and supervised the study; A.A.D., O.I.P., and W.H. per-

formed experiments; A.A.D., O.I.P., and A.S.S. discussed

research results; A.A.D. and A.A.M. wrote the manu-

script; I.B.A. and A.S.S. edited the text of the article;

A.A.D. translated the text into English.

Funding. This work was supported by the Russian

Science Foundation (grant no.23-74-00036 to A.A.M).

Ethics declarations. This work does not con-

tain any studies involving human or animal sub-

jects. Theauthors of this work declare that they have

noconflicts of interest.

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