Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 257-268 © Pleiades Publishing, Ltd., 2024.

257

Safari with an Electron Gun:

Visualization of Protein and Membrane Interactions

in Mitochondria in Natural Environment

Semen V. Nesterov

1,a

*, Konstantin S. Plokhikh

Yuriy M. Chesnokov

Denis A. Mustafin

, Tatyana N. Goleva

, Anton G. Rogov

Raif G. Vasilov

, and Lev S. Yaguzhinsky

National Research Center “Kurchatov Institute,” 123182 Moscow, Russia

Belozersky Research Institute for Physico-Chemical Biology, Lomonosov Moscow State University,

119992 Moscow, Russia

e-mail: semen.v.nesterov@phystech.edu

Received September 24, 2023

Revised February 6, 2024

Accepted February 7, 2024

Abstract— This paper presents new structural data about mitochondria using correlative light and electron mi-

croscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying

biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them

comparable to observing animals in their natural environment on a safari. The paper highlights two areas of

research that can only be accomplished using these methods. The study visualized location of the Aβ42 amy-

loid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in

Alzheimer’s disease. The results showed that the Aβ42 aggregates do not interact with mitochondria, although

some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not direct-

ly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as

sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization

of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the

lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new

mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this

suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-res-

olution tomography expands and complements existing ideas about the structural and functional organization

ofmitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with

each other and with membranes invivo.

DOI: 10.1134/S0006297924020068

Keywords: membrane, mitochondria, oxidative phosphorylation, cryo-electron microscopy, supercomplex, ATP

synthase, respirasome, Aβ42, amyloid aggregates

Abbreviations: ATP,adenosine triphosphate; CLEM,correlative light and electron microscopy; cryo-EM,cryogenic electron

microscopy; cryo-ET, cryogenic electron tomography; OXPHOS, oxidative phosphorylation; TEM, transmission electron

microscopy.

* To whom correspondence should be addressed.

INTRODUCTION

In recent years, cryogenic-electron microscopy

(cryo-EM) techniques have undergone significant im-

provements in both hardware and software. These im-

provements have led to a breakthrough in structural

biology with increased availability and comprehen-

sive image processing capabilities [1-3]. While many

NESTEROV et al.258

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

microscopy studies are investigating isolated proteins

with high resolution, we will narrow our focus on

macromolecular organization of the enzyme systems

in mitochondrial membranes in whole mitochondria

without isolating proteins from them. This article

demonstrates capabilities of the correlative light and

electron microscopy (CLEM) [4]. CLEM, in combination

with genetic engineering, allows visualization of inter-

actions between any proteins of interest, as well as be-

tween proteins and membranes, with a resolution of a

few nanometers. CLEM outperforms solely light-based

techniques in terms of resolution, including super-res-

olution microscopy such as STED microscopy [5]. STED

microscopy allows visualization of membranes but not

individual proteins within them [6]. In this work, we

present our new results, deeply analyze a number of

tomographic data obtained in our previous works, and

discuss the literature devoted to “photoelectron hunt-

ing” of mitochondrial proteins in ‘wild’ natural con-

ditions of their existence in protein–lipid membranes.

Despite the considerable amount of structural

data on mitochondrial proteins, the data on their na-

tive positions in the membrane and matrix are very

limited, which makes it difficult to form an integral

model of the work of these organelles. Recently, cryo-

EM has helped to fill this gap in our understanding.

For example, it has been found that adenosine triphos-

phate (ATP) synthases in the mammalian mitochon-

dria are located at the folds of the inner membrane

(cristae), while components of the respiratory chain

are located in the less curved parts of the membrane.

Additionally, much information has emerged on the

structural interaction of respiratory chain complexes

(respirasome). However, many questions still remain

unanswered. Standard laboratory methods for extract-

ing respirasomes with a mild detergent cannot provide

reliable information on the percentage of respirato-

ry chain complexes that form respirasomes. This is

because some complexes disintegrate during the ex-

traction process, and some could be not extracted from

the membrane. Simultaneously, a detailed analysis of

cryo-EM tomograms enables us to solve this problem.

For instance, we demonstrated that all complexes I

and III are part of respirasomes in the rat heart [7].

Data from numerous scientific groups indicate that

proton transfer between the proton pumps and ATP

synthase occurs not due to electrochemical gradient

between the matrix and intermembrane space vol-

umes, but rather occurs locally with protons moving

laterally along the membrane surface for short dis-

tances. A detailed study of the structure of mitochon-

drial membranes and proteins can provide unique

information necessary for testing existing hypotheses

and modeling proton transfer processes in the oxida-

tive phosphorylation system (OXPHOS) [8]. Additional-

ly, many structural aspects of interaction between mi-

tochondria and other organelles or various aggregates

are still unknown. Even interaction of amyloid aggre-

gates with mitochondrial membrane remains poorly

investigated despite high prevalence of Alzheimer’s

disease. However, colocalization of the fluorescent tags

does not provide sufficient resolution for unambigu-

ous conclusions. In this study, we were able to answer

this question using correlative light-electron microscopy.

MATERIALS AND METHODS

Cultivation of yeast cells. Yarrowia lipolytica Po1f

yeast expressing eGFP-Aβ42 construct [9] were used

in this work. Cells were grown in 250-ml Erlenmeyer

flasks at 28°C on a rotary shaker (220rpm) in 50ml of

semi-synthetic medium containing 1.3% succinate as a

carbon and energy source. Cells were harvested in the

early exponential growth phase (OD

600

=1.0).

Preparation of samples for cryo-EM. Cells were

rinsed with 50 mM phosphate buffer (pH = 5.5) to re-

move the growth medium. Next, they were incubat-

ed with 500 nM MitoTracker Red CMXRos for 30 min.

Afterward, the cells were washed to remove the dye

and concentrated to OD

600

 = 25. To minimize formation

of ice crystals, 5% glycerol was added to the suspen-

sion before plating it on a cryo-EM grid [10]. Electron

microscopy grids underwent hydrophilization using

a Pelco easiGlow unit (USA) at 25 mA current and

0.26 mBar pressure for 30 s. Next, a 5-μl aliquot of cell

suspension was applied to a microscopy grid. Extra liq-

uid from the grids with the test objects was removed

from both sides using filter paper and next grids

were subjected to quick freezing in a liquefied ethane

cooled to liquid nitrogen temperature. When applying

the sample to the grid, temperature in the chamber

of the Thermo Fisher Scientific Vitrobot system (USA)

was maintained at 4°C, and humidity was at least 95%.

If homogenate of cardiac tissue or isolated rat heart

mitochondria were used, a similar grid preparation

procedure was performed, but without the use of glyc-

erol, as described in the previously published works

[7, 11]. Briefly, 3 µl of homogenized Wistar rat heart

tissue or isolated cardiac mitochondria (isolated us-

ing differential centrifugation, without the use of de-

tergents or proteases, as previously described [12])

were applied to a microscopy grid and vitrified using

aVitrobot system.

Fluorescence microscopy. Vitrified yeast samples

were placed in a cryogenic chamber of a Leica THUNDER

Imager EM Cryo CLEM fluorescence microscope (Ger-

many) equipped with a Leica HC PL APO 50×/0.90 CRYO

CLEM objective (Germany). Fluorescence of eGFP-Aβ42

and MitoTracker Red was recorded using appropriate

filters, and colocalization of eGFP-Aβ42 and mitochon-

drial aggregates were assessed. The Leica Thunder

MITOCHONDRIAL PROTEINS AND MEMBRANES IN NATURAL ENVIRONMENT 259

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

algorithm was used to perform 3D reconstruction of

yeast cells, followed by image deconvolution with the

aid of embedded software [13]. Images of the entire

grid provided a “map” for orientation during lamella

etching. Regions of interest were selected based on the

images obtained from the entire grid.

Preparation of lamellae by focused ion beam

(FIB). The vitrified sample on microscopic grids was

transferred to a Thermo Fisher Scientific Versa 3D

scanning electron ion microscope (USA) equipped with

a cryoprobe and a Quorum 3100P liquid nitrogen tem-

perature sample loading system. To reduce charge ac-

cumulation and protect surface from radiation dam-

age during the experiment, a gas-injection system was

used to sputter a protective layer of platinum on the

entire grid surface. Based on the surface morphology

and fluorescence microscopy maps, we selected areas

for preparing thin cell slices using cryo-FIB (Fig. 1).

We have chosen regions closer to the center of the

grid where the cells were arranged in a single layer.

The lamellae were cut in these squares with expec-

tation of passing along a few cells located at a suffi-

cient distance from the metal framework of the grid.

Gallium ions with an energy of 30 keV were used to min-

imize radiation damage to the near-surface layer. The

current was sequentially reduced from 1nA to 30pA.

This approach produced object slices with 150-250nm

thickness. The slice plane was inclined at an angle of

10-12° with respect to the plane of the microscopic grid.

Cryo-EM and cryo-ET. Cryo-EM studies were per-

formed using a Thermo Fisher Scientific Titan Krios

60-300 cryogenic transmission electron microscope

(USA) at accelerating voltage of 300 kV, equipped with

a Falcon II direct electron detection system. For each

microscopic grid, sections were selected to obtain ro-

tating series of images. Each dataset consisted of 56

images taken by tilting the sample between –50° and

60° in 2° angular increments. Data acquisition was

performed automatically using a Thermo Fisher Scien-

tific Tomography 4 software (USA) in low dose mode,

which minimized radiation damage while preserving

native structure of the studied objects. Magnification

of 18,000× (pixel size 0.37 nm and image size 1516 nm)

with a defocus value in the range [–5 μm; –8 μm] was

used to set the tomographic series. Total electron dose

passing through a unit area of the sample during the

entire exposure time did not exceed ~120e

–

/Å

Data processing. Tomographic reconstruction

procedure using the Weighted Back Projections (WBP)

method in the IMOD software package [14] was applied

to the obtained data. Alignment of the rotated series

of cell images was performed by calculating cross-cor-

relation between the regions of the tomographic series

(the “patch tracking” procedure [15]), since it is impos-

sible to introduce colloidal gold nanoparticles into the

sample. In the case of studying fragments of the iso-

lated mitochondria, colloidal gold nanoparticles with

10 nm diameter were used to achieve higher resolu-

tion [7, 16].

In order to reduce noise and resolution anisotro-

py in different planes of the obtained tomograms, fil-

tering in the IsoNet program was used [17]. This tool

uses a U-net neural network and is trained on small

sections of tomograms with addition of extra noise.

Segmentation of mitochondrial membranes was per-

formed in an automatic mode using the program

TomoSegMemTV [18]. Errors in the automatic segmen-

tation were corrected manually using the original to-

mogram data.

The tomograms after filtering were used in man-

ual mode to determine positions of different molecu-

lar complexes. Positions and orientations of various

macromolecules were also determined automatically

using three-dimensional templates of these molecules

in the Dynamo [19] or WARP [20] programs. Subsequent

iterative 3D classification was performed to eliminate

incorrectly selected coordinates. To increase the sig-

nal-to-noise ratio and spatial resolution [21] of mac-

romolecules, averaging of small sections of the tomo-

gram (called subtomograms) containing individual

macromolecules aligned with each other was used [22].

This process, called subtomographic averaging, simul-

taneously provides information about orientation of

macromolecules, which is used for subsequent visual-

ization of the object by inserting a model of the aver-

aged structure in the desired orientation.

RESULTS

Visualization of amyloid aggregates in cells.

Based on fluorescence microscopy data, it has previous-

ly been suggested that the beta-amyloid (Aβ42) aggre-

gates can interact with mitochondrial membranes and

thereby disrupt bioenergetics [9]. For the experiment,

we have chosen a previously used model based on the

aerobically metabolizing yeast Y. lipolytica expressing

the eGFP-Aβ42 construct [9]. It was shown earlier that

expression of Aβ42 or of eGFP-Aβ42 reporter gene con-

struct led to dysfunction and fragmentation of yeast

mitochondria [9]. Colocalization of amyloid aggregates

and mitochondria revealed in this work [9] led to the

hypothesis of direct physical contact of amyloid ag-

gregates with the mitochondrial membrane. To test

this hypothesis, a CLEM microscopy protocol was

performed, culminating in cryo-electron tomography

(cryo-ET) of the selected regions of interest. It is im-

portant to note that cryo-ET is the only suitable meth-

od of investigation for this task, because during the

vitrification process and subsequent microscopic ex-

amination, native structure of the samples is practi-

cally not disturbed, which is an important parameter

NESTEROV et al.260

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

because fluorescent objects are located at a small dis-

tance and even a slight change in their colocalization

due to the use of standard technique of fixing and con-

trasting the sample for electron microscopy could lead

to the appearance of artifacts.

The cryogenic CLEM protocol involved using flu-

orescence microscopy to examine the vitrified sam-

ple for the most representative fields of view where

MitoTracker Red-stained mitochondria and fluorescent

eGFP-Aβ42 aggregates were expected to colocalize.

Once a region of interest was identified, z-stack imag-

ing was performed to reconstruct 3D location of the

areas containing mitochondria and eGFP-Aβ42 aggre-

gates. Based on this information, FIB was adjusted to

obtain a lamella containing the region of interest for

which cryo-electron tomograms were obtained. Fig-

ure1 shows the intermediate results of each stage of

the study.

As a result, a series of detailed tomograms of the

regions of interest were obtained, superimposing both

mitochondrial fluorescence (red channel, MitoTracker

Red) and amyloid aggregates (green channel, eGFP-

Aβ42). At the same time, resolution of the cryo-elec-

tron tomograms was significantly higher than that

of the fluorescence microscope, allowing us to distin-

guish both membranes and individual proteins in the

mitochondria that have characteristic structural fea-

tures, such as ATP synthases (Fig. 1d). The eGFP fluo-

rescence signal allowed to localize amyloid aggregates,

which could not be clearly identified without fluores-

cent labeling and using CLEM. The aggregates were

shown not to interact with the mitochondrial mem-

brane. As can be seen in Fig. 1, some of the aggregates

do not localize near the mitochondria at all, which can

also be seen in the fluorescence microscopy images.

As for the ambiguous areas where the fluorescence

signals overlap, statistical data were collected for sev-

eral dozen of aggregates in these areas (Fig.2) and it

was shown that the vast majority of aggregates were

Fig. 1. Workflow on a cell sample during CLEM microscopy

combined with cryo-ET. a)Photograph of a grid with a vitri-

fied sample under a transmission light microscope. b)Magni-

fied image of the area highlighted in panel(a) with sputtered

platinum layer and an area cut out by cryogenic focused ion

beam method in which there is a thin lamella (highlighted

with a red frame). Scanning electron ion microscopy image.

c) Correlative light and electron microscopy (CLEM) of the

lamella. Overlay of the fluorescence image region correspond-

ing to the etched lamella. Red fluorescence is MitoTracker Red

(mitochondrial marker), green fluorescence is eGFP (from

the eGFP-Aβ42 construct). d) Slice of the tomogram plotted

against the region inside the lamella showing two mitochon-

dria as well as other cell components (cell wall, ribosomes,

amyloid aggregates and various vesicular structures). Inset

shows an enlarged fragment of a mitochondrial crista, with

red arrows pointing at F1 subunits of ATP synthase protrud-

ing from the membrane.

MITOCHONDRIAL PROTEINS AND MEMBRANES IN NATURAL ENVIRONMENT 261

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 2. Visualization and estimation of distances between Aβ42 aggregates and mitochondrial outer membrane on multiple to-

mograms. a)Example of computer reconstruction of a tomogram with automatically labeled mitochondrial membranes (blue),

amyloid aggregates (yellow) and ribosomes (red). b)Distribution of distances between the aggregates and the mitochondrial

outer membrane assembled from the tomographic data.

Fig. 3. Structures of the respiratory chain supercomplex of mitochondria from different organisms. a,c)Different projections of

the respirasome surface structure from Arabidopsis sp. mitochondria obtained by single-particle cryo-EM (resolution about2 Å,

PDBID: 8BPX) [34]. b,d)Different projections of the respirasome structure from rat heart mitochondria obtained by subtomo-

graphic averaging without extraction of the respirasome from the membrane according to [7]. In panels(a), (b), (c) different

complexes within the supercomplex [blue– complexI, purple– complexIII, green– complexIV (only present in rat respira-

somes)] are highlighted by different colors. In panel(c) color mapping is done according to the surface hydrophilicity (blue–

hydrophilic, yellow– hydrophobic) to better illustrate presence of the membrane curvature.

NESTEROV et al.262

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

located at a distance of 30-300 nm from the mitochon-

drial membrane surface.

The closest aggregates had no physical contacts

with the mitochondrial membrane. This finding is

important for understanding the mechanisms of Alz-

heimer’s disease development, as it suggests that the

disturbance in mitochondrial bioenergetics that cor-

relates with aggregate accumulation [23, 24] is not

caused by the direct effect of amyloid aggregates on

mitochondrial structure and function, but is mediated

by other factors.

Study of the topology of the inner mitochondri-

al membrane and proteins of the OXPHOS system

within it. The single-particle cryo-EM method, based

on computer classification and averaging of huge ar-

rays of images obtained by transmission electron mi-

croscopy(TEM), allows us to study structures of large

protein complexes and supercomplexes. Presence or

absence of supercomplexes has long been a contro-

versial topic in bioenergetics, yet this fact is extremely

important. Presence of supercomplexes implies direct

transfer of metabolites between them with minimal

involvement of diffusion in this process. Such arrange-

ment is much more resistant to stress, reduces electron

leakage and ROS generation, making the whole system

more energy efficient. Existence of large respiratory

chain supercomplexes in mitochondria prior to the

use of cryo-EM was demonstrated by the presence of

common migration of their components during native

electrophoresis after protein extraction from mem-

branes with mild detergents [25]. Nowadays, existence

of the respiratory supercomplexes– respirasomes – is

no longer in doubt due to cryo-EM imaging of single

particles, as their structures from different organisms

and tissues have been demonstrated in a large number

of studies [26-33]. Nevertheless, more detailed issues,

such as exact configuration and composition of super-

complexes under certain conditions, their functional,

regulatory and structural significance for bioenerget-

ics, remain controversial. In particular, mechanisms of

regulation by respirasomes, existence of any structur-

al interaction between them and ATP synthases, and

role of the membrane in these processes are not fully

understood. To address these issues, we analyzed the

data obtained in our previous studies together with

the new literature data. Figure 3 shows structures of

the respirasomes from the evolutionarily distant Ara-

bidopsissp. mitochondria and rat heart mitochondria,

which surprisingly turned out to be not only similar in

structure, but also to have a similar curvature of the

membrane, creating its kink at the point of contact of

complexesI andIII.

Cryo-ET (cryo-electron tomography) allows us to

reconstruct entire membrane regions by determining

location of the proteins with characteristic structur-

al features. Such proteins include ATP synthases and

complexesI, which have unique shape of a large hydro-

philic part protruding from the membrane (Fig. 4, a, b).

In addition, it is possible to reconstruct three-dimen-

sional structure of the inner mitochondrial membrane

with resolution that significantly exceeds that of to-

mography, based on analysis of the slices of fixed mi-

tochondrial samples. As a result, cryo-ET shows the

inner membranes of cardiac mitochondria in greater

detail and are not represented as wave-like lamellar

structures, as interpreted from the TEM ultrathin sec-

tions [35], but as a complex structure of many small

flat fragments interspersed with many tubular junc-

tions (Fig. 4). This structure answers the question of

how ATP synthase dimers are arranged in the inner

membrane of cardiac mitochondria. The most abun-

dant oligomers of ATP synthases are found at the

bends of the highly curved cristae [36], but there are

fewer long clusters of ATP synthases in the regions

with lower curvature, where a less ordered structure

is created in which dense packing of respirasomes

and ATP synthases is impossible (Fig.4b, right row of

ATP synthases).

On a smaller scale (without visualization of indi-

vidual membrane proteins), it is possible to perform a

detailed reconstruction of the inner membrane of car-

diac mitochondria using high resolution images gener-

ated with cryo-ET. We have recently performed such

reconstruction to visualize position of the large dehy-

drogenase complexes relative to the membrane under

natural conditions [11]. The obtained data suggest

(Fig. 4) that the cardiac mitochondria are a complex

maze of highly curved membranes with a lamellar-

tubular structure. At the same time, ATP synthase di-

mers cannot be located in the completely lamellar flat

areas [36]. From the point of view of ensuring optimal

structure and functionality of mitochondria, the lamel-

lar structure of the membrane seems to be unsuitable

for accommodating the key components of OXPHOS.

This issue can be partially resolved by formation of the

wave-like curved structures of the inner membrane bi-

layer, on the curved areas of which ATP synthases can

be localized. Such topology has been indeed observed

in some TEM images [37]. However, the membrane

structures are often visualized as lamellar, which pre-

vents formation even of the strained structures with

ATP synthase dimers, one of which is shown in Fig.4b.

The paradox of the presence of lamellae visible by

classical electron microscopy was resolved by using

higher resolution of cryo-ET. The computer recon-

struction showed that toroidal holes are formed in the

lamellar regions at the edges of which ATP synthase di-

mers could be located, and close to them in the regions

with less curvature – respirasomes. In contrast to the

cristae edges shown in Fig.3, the toroidal openings at

the edges could accommodate not only one row of ATP

synthase dimers, but even two rows of the tightly at-

MITOCHONDRIAL PROTEINS AND MEMBRANES IN NATURAL ENVIRONMENT 263

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 4. Reconstruction of the complex structure of the mitochondrial membranes of the heart. a)Reconstruction of a tomogram

of a large area in a whole intact mitochondrion of the rat heart. Blue– cores of ketoglutarate dehydrogenase or branched-chain

ketoacid dehydrogenase complexes, green– cores of pyruvate dehydrogenase complexes. b,d)Fragments illustrating presence

of holes of toroidal topology. c)Reconstruction of a fragment of a tomogram of a rat heart mitochondrial crista. ATP synthases

are shown in yellow, complexesI in blue, dimers of complexesIII in purple and complexesIV in green. The membrane shown

in grey is not transparent and covers hydrophobic parts of the complexes. e)Thin section of part of the crista at the location

indicated by the arrows in panel(c). The membrane is shown transparent, allowing visualization of the hydrophobic parts of

thecomplexes. The image created based on the tomographic data obtained in the course of previous work [7, 11].

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Fig. 5. Schematic model of putative location of protein complexes of the OXPHOS system in the vicinity of toroidal holes in the

pseudolamellar regions of cristae. The model was constructed based on manual processing of cryo-ET images of several toroidal

holes with labelling of the location of F1 subunits of ATP synthases in the inner membrane of rat heart mitochondria. Yellow

indicates ATP synthase dimers, blue indicates respirasomes, dark grey indicates membrane and light grey indicates intermem-

brane space.

tached ATP synthases. This ensures the most efficient

use of the rather small (compared to the total area of

the cristae) curved regions of the membrane for locat-

ing ATP synthase dimers on them. An oligomeric struc-

ture of the ATP synthase dimers similar to that shown

in Fig.4 has been observed in the toroidal holes in the

region with high curvature. However, the preliminary

manual analysis shows a larger number of structures

in some of the toroidal holes that correspond in size to

the F1 subunits of ATP synthases, suggesting that the

two rows of ATP synthase dimers may be present at

the membrane fold at the same time. Due to the high

protein density, which prevents high resolution, accu-

rate computer reconstruction using averaging of ATP

synthases in these regions has not yet been possible.

Schematic representation of the proposed arrange-

ment of the OXPHOS complexes in such structures is

shown in Fig.5.

This complex topology of mitochondrial mem-

branes may be maintained by high concentration of

the cone-shaped lipids such as cardiolipin and phos-

phatidylethanolamine in the membrane. Presumably,

the membrane junctions are so enriched in these lip-

ids, and their structure is so different from the bilayer,

that they have been defined by NMR as a non-bilayer

lipid phase [37, 38]. Given the necessity of cardiolipin

for operation of the OXPHOS system [39] and optimiza-

tion of its structure to ensure proton transport across

the membrane [8], we can assume that the propensity

of respirasomes and ATP synthases to generate mem-

brane curvature may, at least in part, be evolutionarily

determined by the need for cardiolipin concentration

at the interface of OXPHOS system proteins.

DISCUSSION

Lack of direct interaction of amyloid aggre-

gates with mitochondrial membranes. Advanced

models of chronic diseases associated with mitochon-

drial dysfunction, based on the aerobic metabolic yeast

Y. lipolytica [9], have proven to be a powerful tool to

reveal intracellular actions of the markers of these dis-

eases, which is an important task in the case of pathol-

ogies associated with mitochondrial dysfunction [40],

since mitochondria are quite deeply involved in the

cell signaling systems, and it is often difficult to under-

stand whether their dysfunction is primary or medi-

ated by pathological changes in the cellular environ-

ment. The yeast Y. lipolytica, as a unicellular organism,

lacks complex system of intercellular interactions and,

in addition, possesses a branched system of fully func-

tional “animal-type” mitochondria containing all the

complexes of the respiratory chain.

It was shown [9] that the Aβ42 expression in the

yeast caused mitochondrial dysfunction, which is char-

acteristic for the neurons at the early stages of Alzhei-

mer’s disease development. In addition, colocalization

of mitochondria and amyloid aggregates suggested the

presence of physical contact between the mitochondria

and amyloid aggregates, as assumed in some other work

[41]. Since vitrification of the samples for cryogenic mi-

croscopy is the least destructive method of object fixa-

tion, the data obtained in this work could be considered

as the most reliable showing absence of the contact be-

tween the amyloid protein aggregates and mitochondria.

Nevertheless, distribution of the distances between

the aggregates and mitochondrial membrane indicates

MITOCHONDRIAL PROTEINS AND MEMBRANES IN NATURAL ENVIRONMENT 265

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

concentration of aggregates near mitochondria, which

may be essential for the processes of mitochondrial

substrate transport or for the effects of aggregates on

the protein biosynthesis due to the large number of ri-

bosomes also located near mitochondria.

Model of topological coupling of supercomplex-

es in the mitochondrial membrane. An interesting

peculiarity that was identified owing to cryo-ET was

the fact that the ATP synthases not only dimerize (this

can be detected by native electrophoresis), but also form

oligomeric arrays that deform the membrane and in-

duce a significant curvature on it [16, 42, 43]. It turns

out that respirasomes also affect membrane curvature,

as shown in the cryo-EM images of respirasomes from

different organisms [30, 34]. Furthermore, interaction

of the complex I and complex III dimers in respira-

some is conserved, and structure of the respirasome is

highly similar between the species [44] suggesting that

interaction of the respirasome with the membrane is

also conserved. This implies that association of respi-

rasomes with ATP synthases shown in [7] (Fig. 4c)

could be achieved largely through the mechanical prop-

erties of the membrane, which tend to compensate for

excessive tension. The ATP synthase dimers create

a region of maximum membrane curvature close to

them [36], thereby creating tension in the bilayer and

distorting it from the energetically optimal structure

that would be present if the lipids self-assembled in

the absence of the protein. In the region away from

ATP synthases, the membrane tends to approach the

equilibrium topology of a lamellar bilayer. At the same

time, respirasomes also generate membrane tension,

but the lipid bilayer around them has much less cur-

vature than near the ATP synthase dimers. In order

for respirasomes not to create a separate region of

membrane curvature, it is advantageous for them to

be located closer to ATP synthases in the part of the

membrane where the curvature is already optimal for

them and no additional energy is required to mechani-

cally distort the lipid bilayer. In such case, preferential

orientation of respirasomes along one axis should be

observed, which is indeed confirmed by the experi-

ments with cardiac mitochondria [7]. Thus, the force

attracting respirasomes to ATP synthase has an entro-

pic topological nature and is determined by the mem-

brane, which tends to minimize its deviation from the

approximate equilibrium bilayer structure. It should

also be noted that the integral protein complexes that

create curvature of the mitochondrial membrane also

create around themselves membrane domains (rafts)

of cardiolipin and other lipids that have conical shape,

since they are the ones best suited to create curvature

in the lipid bilayer [45]. Binding of ATP synthases and

respiratory complexes (both individually and assem-

bled into respirasomes) to cardiolipin has been repeat-

edly confirmed in experiments [38, 46-53]. Ability of

cardiolipin to form membrane domains has also been

experimentally demonstrated [54].

Sorting of the proteins based on their interaction

with membrane lipids is well known – such sorting

system based on rafts and nanorafts operates in the

cell [55]. Sorting of the proteins in membrane rafts

is mainly based on coincidence or mismatch of the

thickness of their hydrophobic membrane zone with

the thickness of a certain cluster of membrane lipids

(hydrophobic mismatch principle). The above data on

mitochondrial membranes allow us to add to the well-

known principle of clustering of proteins with similar

hydrophobic zone another sorting principle based on

affinity of the protein complexes to the zones of differ-

ent membrane curvature. Complex topology of the in-

ner membrane of cardiac mitochondria and presence

of the non-bilayer phases in it suggest that self-orga-

nization of the OXPHOS system complexes in the car-

diac mitochondria could proceed via the mechanism

similar to the in meso crystallization of membrane

proteins[56].

Thus, the above data suggest that the structure

of respirasomes is evolutionarily selected to ensure

localization of respirasomes close to ATP synthase di-

mers and thus clustering of the entire OXPHOS system

through interaction with the membrane lipids.

CONCLUSION

This work demonstrates experimental application

of one of the most advanced methods in structural bi-

ology to study interaction of protein aggregates with

membranes, topology of the membranes themselves,

and mutual arrangement of membrane proteins in mi-

tochondria under native conditions. A series of new

experiments using CLEM have shown that amyloid

aggregates do not interact with the outer mitochon-

drial membrane and do not enter the mitochondria.

In addition, our own recent data and recent literature

data obtained using cryogenic-electron transmission

microscopy and tomography of mitochondria and mi-

tochondrial membranes have been analyzed in more

detail. Complex arrangement and intricate topology of

the rat heart mitochondrial membranes, enriched in

large supercomplexes that influence membrane cur-

vature, are shown. In complete agreement with this, it

was shown that the cardiac mitochondrial membrane

has only small regions where it is close to a flat bilayer,

whereas most of the membrane is a complex network

of highly curved junctions on which rows of ATP syn-

thase dimers and respiratory chain supercomplexes

are located. The regions of the membrane that appear

lamellar on the TEM thin sections are actually dotted

with a multitude of holes containing tightly packed

clusters of protein complexes of the OXPHOS system.

NESTEROV et al.266

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

This paper also discusses theoretical framework that

explains clustering of the membrane proteins of the

OXPHOS system and cardiolipin by minimization of

the strain energy of the lipid part of the membrane.

This principle of membrane protein organisation is

proposed to be termed topological.

Contributions. S.V.N. planned and performed ex-

periments, manually analyzed tomograms, prepared

the manuscript; D.A.M., T.N.G., and A.G.R. planned and

conducted CLEM experiments with yeast; K.S.P. and

Yu.M.Ch. conduct TEM and CLEM experiments, perform

computer processing of the tomographic data. L.S.Y.

and R.G.V. developed the study concept, supervised the

study. All authors discussed the results and contribute

to the manuscript editing.

Funding. This work was financially supported

by the NRC Kurchatov Institute (thematic plan 1f.4.1

“Study of energy generation, transfer and distribution

processes in living organisms aimed at finding new

approaches to the development of therapeutic agents,

new bioenergetic devices and artificial photosynthesis

systems”).

Ethics declarations. This work does not contain

any studies involving human and animal subjects.

The authors of this work declare that they have no

conflicts of interest.

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