Available online at www.sciencedirect.
com
ScienceDirect
Biophysical and molecular mechanisms of ESCRT
functions, and their implications for disease
Simona Maria Migliano1,2,a, Eva Maria Wenzel1,2,a and
Harald Stenmark1,2
Abstract
The endosomal sorting complex required for transport
(ESCRT) machinery evolved early in evolution to sculpt and cut
cellular membranes. Consisting of three subcomplexes termed
ESCRT-I, -II and -III, this machinery is recruited to various
cellular locations to perform key steps in essential processes
such as protein degradation, cell division, and membrane
sealing. Here we review recent discoveries that have shed light
on biophysical and molecular mechanisms of ESCRTs in
endolysosomal protein degradation and nuclear envelope
sealing, and we discuss how dysfunctional ESCRTs can lead
to diseases such as cancer and neurodegenerative disorders.
Addresses
1
Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine,
Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo,
Norway
2
Department of Molecular Cell Biology, Institute for Cancer Research,
Oslo University Hospital, Montebello, N-0379 Oslo, Norway
Corresponding author: Stenmark, Harald (stenmark@ulrik.uio.no)
a
These authors made equal contributions.
Current Opinion in Cell Biology 2022, 75:102062
This review comes from a themed issue on Membrane Trafficking
2022
Edited by Chiara Zurzolo and Benjamin Glick
For complete overview of the section, please refer the article collection -
Membrane Trafficking 2022
Available online 3 March 2022
https://doi.org/10.1016/j.ceb.2022.01.007
0955-0674/© 2022 The Author(s). Published by Elsevier Ltd. This is an
open access ar ticle under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Cellular membranes undergo continuous dynamic
remodelling, such as tubulation, budding, fission and
fusion events. Some of these membrane reformation
processes are mediated by the ESCRT machinery, in
particular when the deformation and scission occurs in a
topology away from the cytosol. In contrast to classical
scission machineries, such as clathrin or COPI/II medi-
ated vesicle formation, the ESCRT machinery is capable
of reshaping lipid membranes without acting as a protein
scaffold. Instead, it is able to cut membranes from the
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luminal side. The core machinery consists of three
subcomplexes termed ESCRT-I, -II and -III, which can
be complemented by accessory proteins providing energy
(the AAA ATPase VPS4) or specificity of recruitment
(such as ESCRT-0, see below). The main task of this core
machinery is to assemble at preexisting membrane stalks
to constrict and abscise them, or to seal holes in mem-
branes, for example when the plasma membrane or
membranes of cell organelles are punctured (reviewed in
Refs. [1,2]). In recent years, more and more cellular
functions have been discovered, ranging from the for-
mation of intraluminal vesicles in endosomes, via cell
division and virus budding to membrane repair and
sealing processes of variable size scales. The multitude of
ESCRT functions are reviewed in comprehensive recent
reviews [3e6]. In the present review we will focus on the
molecular and biophysical mechanisms of the ESCRT
machinery, highlighting their functions at multivesicular
endosomes and nuclear and micronuclear envelopes
(Figures 1 and 2). Further, we will discuss the role of
ESCRTs in diseases (Figure 3).
Structural and biophysical insights into the
membrane remodeling activity of the
ESCRT machinery
Thanks to the concerted efforts from in vivo, in vitro and in
silico studies, significant progress in our mechanistic un-
derstanding on how the ESCRT machinery remodels
membranes has been made during the last few years. We
will start by summarizing the current mechanistic insight
using the most classical ESCRT function, the formation of
intraluminal endosomal vesicles, as an example. We will
hereby focus on mammalian cells and yeast, which contain
ESCRT-0, -I, -II and III. Although the HRS-STAM-
containing ESCRT-0 is conserved from budding yeast to
man, there are organisms that can do without this com-
plex. Bacteria and archaea express ESCRT-III or ESCRT-
III like proteins but lack overt ESCRT-0, -I and eII
components [7e9]. This obviously reflects the involve-
ment of ESCRT-III in non-endosomal functions in these
organisms. More surprisingly, HRS and STAM are not
found in plants, whereas ESCRT-I, -II and eIII are
conserved. However, there are indeed functional ESCRT-
0 components in plants, including TOM1-like proteins
that share the functional domains of HRS and STAM
[10,11] and the PtdIns3P-binding protein FREE1 [12].
Current Opinion in Cell Biology 2022, 75:102062
2 Membrane Trafficking 2022
Figure 1
ESCRTs constitute a multi-protein machinery that controls a wide
variety of cellular processes. Mutations or loss of essential ESCRT
components have fatal consequences on cellular function and can lead to
severe pathologies. In this review we highlight the dynamics and structure
of ESCRT proteins and their importance in endosome maturation, at the
nuclear and micronuclear envelope and in diseases. The ESCRT ma-
chinery thus displays a high modularity so that the components can be
flexibly matched across organisms and membrane remodeling functions.
The ESCRT machinery thus displays a mix-and-match
type of versatility across organisms and membrane
remodelling functions.
Formation of intraluminal endosomal vesicles
For the internalization and subsequent degradation of
activated transmembrane receptors the formation of
intraluminal vesicles resulting in multivesicular endo-
somes is a prerequisite. Ubiquitinated cargo molecules
on the limiting membrane of endosomes are recognized
by ESCRT-0, which consists of HRS and STAM.
Membrane recruitment of ESCRT-0 is promoted by
binding of HRS to the endosomal lipid, phosphatidyl-
inositol 3-phosphate (PtdIns3P) [13]. ESCRT-0 in turn
recruits ESCRT-I/-II and clathrin, leading to the for-
mation of microdomains on the endosomal limiting
membrane [14e16].
The next step after initial target recognition and clus-
tering is the deformation of the endosomal membrane to
form negatively curved pits [17]. The likely mechanism
for this is protein crowding [18e20] of upstream
ESCRTs together with transmembrane cargo molecules
and clathrin. Based on mathematical modeling and
electron microscopy imaging of ESCRT-III depleted
cells, this initial membrane deformation, surprisingly,
neither requires external energy (ATP), nor the pres-
ence of ESCRT-III subunits [21]. A definitive role of
protein crowding in ESCRT-mediated membrane
Current Opinion in Cell Biology 2022, 75:102062
deformation will have to await in vitro reconstitution
experiments specifically designed to address this issue.
Thus, the upstream ESCRT machinery or other recruiting
factors such as viral GAG proteins [22] initiate membrane
deformation and allow polymerization of the downstream
ESCRT machinery during processes lacking pre-existing
membrane stalks or holes. ESCRT-III components are
recruited by ESCRT-I/-II and/or Bro domain proteins [23]
and preferentially polymerize at curved membranes
[24e27]. The polymerization of ESCRT-III subunits can
occur in various conformations of the individual subunits
(closed, open or intermediate conformation), often
forming copolymers with other ESCRT-III subunits. The
resulting morphologies as circles and spirals are by now
well characterized through structural biology approaches,
cryo-electron microscopy and atomic-force microscopy
[28e30]. However, recent structural data have unraveled
that also the ESCRT-I subcomplex can form helical fila-
ments [31], underpinning that the upstream ESCRT
machinery might be mechanistically more important for
membrane deformation and ESCRT polymerization than
previously anticipated. Indeed, research in the last few
years has been heavily focused on understanding
constriction and scission of membrane necks mediated by
the ESCRT-III machinery and the field has seen a
tremendous gain of knowledge on how membrane
constriction and scission is mediated by the downstream
ESCRT machinery (reviewed in Refs. [32e34]). The
structures, molecular properties and interplay of various
ESCRT-III filaments has been investigated by elegant
in vitro reconstitution studies using purified ESCRT sub-
units [27,35e40]. These detailed studies have culmi-
nated in a unifying model which reconciles buckling/
unbuckling processes and protomer conversion elements,
which in combination lead to constriction and scission of
narrow membrane necks [40]: Following nucleation by the
upstream ESCRT machinery, CHMP4/Snf7 polymerizes,
followed by sequential and ordered recruitment of
CHMP3/Vps24 and CHMP2/Vps2 and finally CHMP1/
Did2 and IST1/Ist1. The slightly varying biophysical
properties of each ESCRT-III subunit and their coordi-
nated assembly and disassembly lead to a continuous
constriction of the forming membrane neck. This dynamic
turnover is regulated by the AAA ATPase VPS4/Vps4.
VPS4/Vps4 binds via its “microtubule interacting and
trafficking” (MIT) domain the carboxy-terminal “MIT
interaction motif” (MIM) domains of most ESCRT-III
subunits and mediates their extraction and exchange,
which is a prerequisite for successful narrowing of the
neck [41,42]. Scission will presumably occur through
hemifusion as soon as a critical membrane distance below
3 nm is reached [43]. Exactly how ESCRT-III polymers
reach such a close distance is not yet completely under-
stood, since the tightest observed structure is 4.4 nm
formed by IST1-Chmp1b [39].
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 ESCRTs - mechanisms and implications for disease Migliano et al. 3

Figure 2

Coordinated assembly and disassembly of ESCRT proteins at (a) endosomal and (b) (micro)nuclear membranes. (a) ESCRT proteins recognize
ubiquitinated receptors at the endosomal surface. A finely tuned and dynamic assembly of clathrin, ESCRTs and accessory proteins sequester ubiq-
uitinated receptors in microdomains and package these in intraluminal vesicles (ILVs). (b) Nuclear envelope rupture and reformation during cytokinesis
recruits ESCRT components to the nuclear membranes. While a coordinated membrane repair occurs at the primary nucleus, an extensive influx of
CHMP7 leads to excessive CHMP7-LEMD2 and unrestrained ESCRT assemblies in micronuclei. This results in an uncontrolled membrane remodeling
and a micronuclear catastrophe.

ESCRT functions at nuclear and micronuclear
envelopes
During open mitosis, the nuclear envelope disassembles
in prophase in order to allow alignment of the duplicated
chromosomes in metaphase and their separation in
anaphase. When the envelopes of the daughter nuclei
reform during late anaphase and telophase, they are
traversed by spindle microtubules, which need to be
removed to allow formation of intact daughter nuclei.
ESCRT-III proteins play two important roles during this
process. Firstly, they assemble at the microtubule-
traversed holes in the nuclear envelope to recruit the
microtubule-severing enzyme Spastin [44]. Secondly,
when Spastin has severed the microtubule bundles,
ESCRT-III mediates closure of the remaining hole in
the double-membrane nuclear envelope by a mecha-
nism that is topologically related to ILV biogen-
esis [44,45].
Central in recruitment of ESCRT-III to holes in the
nuclear envelope is CHMP7, a protein with an ESCRT-
II-like N-terminus and an ESCRT-III-like C-terminus
[44]. CHMP7 is actively transported out of the intact
nucleus by Exportin 1, but when the nuclear envelope is
disassembled or damaged, CHMP7 associates with
chromatin via the inner nuclear envelope protein LEM2,
which in turn binds to the chromatin-associated protein
BAF [46e48]. At least in budding yeast, the association
of CHMP7 with LEM2 requires binding of CHMP7 to
phosphatidic acid at nuclear envelope herniations [49].
Recent evidence indicates that CHMP7 exists in an
autoinhibited conformation in the absence of LEM2,
and that LEM2 binding triggers an opening that causes
CHMP7 and LEM2 to co-polymerise around microtu-
bules at the nuclear envelope interface. This polymer-
ization leads to formation of phase-separated
condensates via a low-complexity region of LEM2,
which effectively function as O-ring seals [48]. In this
way, leakage of nuclear components is prevented while
Spastin and ESCRT-III perform their jobs in microtu-
bule severing and membrane sealing. But how is the
association between CHMP7 and LEM2 prevented

www.sciencedirect.com Current Opinion in Cell Biology 2022, 75:102062

4 Membrane Trafficking 2022
Figure 3
Mutations or loss of essential ESCRT proteins can lead to severe pathologies. ESCRTs play a central role for endosomal maturation and trafficking,
as well as other cellular processes and virus replication and release. Defects and irregularities in ESCRT assemblies can therefore substantially influence
tissue homeostasis and organ integrity during development and adulthood and lead to the development of severe pathologies (Created with BioRender.
com).
when the entire nuclear envelope is disassembled in
prophase? This is achieved by the mitotic kinase CDK1,
which phosphorylates CHMP7 at mitotic entry. Phos-
phorylation renders CHMP7 unable to interact with
LEM2, and local dephosphorylation at the nascent nu-
clear envelope licences the LEM2-CHMP7 interaction
that triggers ESCRT-III recruitment [50].
The function of ESCRT-III in nuclear envelope sealing
is important during normal cell division and after
damage to the nuclear envelope. However, there is also
an example that ESCRT-III recruitment can be harmful
to cells. This occurs when ESCRT-III is recruited to the
envelopes that enclose micronuclei, small nuclear-like
structures that contain a single chromosome or chro-
mosome fragment. ESCRT-III is not normally recruited
to micronuclei, but when they occasionally break, this
triggers recruitment of LEM2, CHMP7 and ESCRT-III
by similar mechanisms as those employed by primary
nuclei [47]. Whereas ESCRT-III recruitment to holes in
the primary nucleus is limited and transient, ESCRT-
III, CHMP7 and LEM2 recruitment to holes in micro-
nuclei is excessive and long-lasting, leading to collapse
of the micronucleus and massive damage to the chro-
matin it contains. This is similar to a phenomenon
known as chromosome shattering or chromothripsis, a
condition strongly associated with cancer progression.
Why does ESCRT-III recruitment to ruptured micro-
nuclear envelopes run out of control? Mathematical
Current Opinion in Cell Biology 2022, 75:102062
simulations suggest that the differential ESCRT-III
recruitment to nuclei and micronuclei is related to
their size. LEM2, CHMP7 and ESCRT-III recruitment
to primary nuclei is restricted to discrete holes, shows
limited lateral diffusion, and is reversible as soon as the
hole is sealed. In contrast, when these molecules are
recruited to holes in micronuclei, the lateral diffusion
becomes a problem because of the small area of the
micronuclear envelope, leading to unrestricted recruit-
ment and micronuclear catastrophe [47].
The diverse roles of ESCRTs in diseases
Loss or mutations in essential components of the
ESCRT protein machinery and resulting missorting of
endocytic cargo can lead to severe pathologies, many of
which affect the nervous system [6]. For instance, a
depletion of HRS and TSG101 lead to missorting of M2
acetylcholine receptor and loss of PTPN23 disrupts
neurotrophin receptors sorting. This in turn has nega-
tive effects on neurite outgrowth, dendritic branching
and neuronal development and is critically linked to
diseases such as Alzheimer’s, Parkinson’s, schizophrenia
and addiction [51e53].
Similar pathologies have recently been associated with
mutations in UBAP1 (ESCRT-I), CHMP2B (ESCRT-
III) and PTPN23 (interacting with ESCRT-0, -I, -III)
[54e57]. Neurons seem to be particularly susceptible to
mutant CHMP2B, which is also found tightly associated
with Frontotemporal Dementia [58]. At the cellular
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 ESCRTs - mechanisms and implications for disease Migliano et al.
level, all these pathologies have an abnormal (mostly
enlarged) endosome morphology in common, which is a
consequence of defective cargo degradation or recycling.
Missense mutations in the ESCRT-III-regulatory
ATPase VPS4A have been related to Congenital Dyser-
ythropoietic Anemia, a disease which displays defects in
cytokinesis and abscission of erythroblasts [59]. Simi-
larly, patients with de novo mutations in the catalytic
central pore of the hexameric VPS4, display structural
abnormalities in the brain, as well as visual and auditory
dysfunctions [60]. Mutations in VPS4 and ESCRT-III
are also known to affect nuclear envelope structure
[6]. New findings underline the known interactions
between the chromosomal scaffold protein SATB2, the
inner nuclear membrane protein LEMD2, and the
ESCRT-III/VPS4 machinery. These interactions are
required for synaptic activity-triggered nuclear mem-
brane remodeling in pyramidal neurons, a process which
is thought to be affected in cognitive dysfunction and
schizophrenia [61].
Like neurological pathologies, many cancers are associ-
ated with mutation or loss of essential ESCRT proteins,
leading to aberrant receptor trafficking and enhanced
cell proliferation or migration. For example VPS28 in
ESCRT-I is involved in the trafficking and secretion of
Awd, the Drosophila homolog of the metastatic sup-
pressor NME1/2 [62]. Similarly, a loss of the ESCRT-I
subunit VPS37A/HCRP1 leads to an upregulation of
the immune checkpoint ligand PD-L1 and to an in-
crease in cell migration [63].
Exciting advances have been made to investigate
ESCRT proteins and immune responses. Especially
ESCRT-mediated extracellular vesicle formation and its
role in antigen delivery is well established [64,65]. Along
these lines, the critical regulation of CD40, a receptor
responsible for B cell development, has been found to
depend on the ESCRT-II subunit VPS25 and the
ESCRT-III related CHMP5. Failures in transport and
lysosomal degradation of CD40 increase the risk of
developing autoimmunity, immunodeficiency and
cancer [66]. Interestingly, also a new connection be-
tween VPS4 and inflammatory responses has been
discovered. Whereas disruption of normal VPS4B func-
tion has been shown to correlate with cancer, synthetic
lethality of the VPS4A and VPS4B paralogs can also
induce an inflammatory response leading to cell death.
These findings could allow a novel therapeutic strategy
for these cancer patients, by developing a specific VPS4
inhibitor [67].
Immune responses are also required in terms of virus
infection. It is well established that ESCRT proteins are
hijacked by various types of viruses to facilitate their
replication and egress [68]. In recent years, more and
more virus types have been added to this list, such as
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5
recently shown for Chikungunya, Vaccinia and human
Cytomegalovirus [69e71]. Interestingly, some ESCRTs
can also suppress viral replication. This has been found
for HRS (ESCRT-0), which interacts with the viral
antisense protein APH-2 in Human T Cell Leukemia
Virus Type 2 (HTLV-2) infected cells [72].
Outlook
Since their discovery, the field of ESCRT protein
research has continuously expanded and an abundance
of ESCRT driven processes, as well as ESCRT-
interacting proteins has been described. Extensive ef-
forts have been made in understanding the structure
and dynamic behavior of ESCRT assemblies in different
cellular processes, all highlighting the well-orchestrated
nature of ESCRT proteins. Point mutations and small
differences in ESCRT protein expression levels can
have severe consequences for organelle and cellular
function, tissue integrity and even lead to fatal patholo-
gies. Although a large part of ESCRT driven processes
has already been investigated, it will be exciting to un-
cover their exact molecular mechanisms, especially
related to ESCRT recruitment to specific intracellular
sites. It will also be interesting to learn which pathogens
are capable of hijacking ESCRT proteins for their pur-
poses and how this ancestral membrane deformation
machinery has evolved and been perfected over time.
Conflict of interest statement
Nothing declared.
Acknowledgements
The authors would like to thank the Norwegian Cancer Society (grant no.
182698), the South-Eastern Norway Regional Health Authority (grant no.
2018081), the Research Council of Norway (grant nos. 262652, 302994),
the European Research Council (grant no. 788954) and Trond Paulsen and
the Radium Hospital Foundation (InvaCell grant) for financial support.
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