Cell Tissue Res (2000) 301:5–17
Digital Object Identifier (DOI) 10.1007/s004410000193
REVIEW
Georg Häcker
The morphology of apoptosis
Received: 7 January 2000 / Accepted: 28 January 2000 / Published online: 30 March 2000
Abstract The concept of apoptotic cell death as an es-
sential part of the development and life of complex or-
ganisms has been devised in different situations and test-
ed from various angles. This review article discusses the
morphological changes during death by apoptosis. In
cells undergoing apoptosis, an intracellular signalling
pathway operates cell autonomously to implement the
death and disposal of the cell. The similarity of the bio-
chemical events during apoptosis in different situations
is reflected by a high uniformity of morphological
changes in many situations of naturally occurring or ex-
perimentally induced cell death. The unifying concept of
apoptosis has been derived from the observation of this
morphological consistency of dying cells almost 30 years
ago. Since then, we have learned much about the intra-
cellular signalling in the apoptotic process and the mo-
lecular background has been delineated which guides the
initiation of the morphological changes. Here, an attempt
is made to present the current knowledge about the mo-
lecular events in the development of these morphological
alterations and to place these changes in the context of
apoptotic cell death.
Key words Apoptosis · Morphology · Caspases
Introduction
Cell death is an integral part of the normal life of com-
plex organisms, and it has been observed for more than a
century in various situations (for review, see Clarke and
Clarke 1996). The first description noted only the fact
that cells disappear during normal development in a toad
species (Vogt 1842). A large number of the early mor-
phological studies describe the visible changes of cell
G. Häcker (✉)
Institute for Medical Microbiology,
Technische Universität München, Trogerstrasse 9,
81675 Munich, Germany
e-mail: hacker@lrz.tum.de
Tel: +49 89 4140 4121, Fax: +49 89 4140 4868
© Springer-Verlag 2000
death both in vivo and in vitro, and the morphological
changes and the disappearance of cells have for some
time been the only indication of cell death (Clarke and
Clarke 1996).
It was on morphological grounds that Kerr et al. in
1972 developed the unifying concept of a form of cell
death as an instrument for the disposal of unwanted cells
during embryonic development, during normal cell turn-
over in proliferating tissues and in pathological situa-
tions (Kerr et al. 1972). The term ‘apoptosis’ for this
form of cell death was suggested by these investigators
and has since found wide acceptance. The excellent mor-
phological description and thorough observations by
Kerr et al. and in earlier work (for review see Kerr et al.
1972) – now supplemented by some more recently de-
fined features of apoptosis – are still a reliable basis for
the detection of apoptosis. Apoptosis-associated morpho-
logical changes have provided the background for the
definition of apoptosis-inducing stimuli and to guide the
investigation of the biochemistry and signal transduction
of apoptosis. Today we know that the morphological
changes are the result of the activation of an intracellular
signal transduction pathway, probably the only function
of which is to kill the cell and to organise the disposal of
the body.
Names and forms of cell death
A number of alternative expressions have been coined
over this period to describe cell death, such as degenera-
tion, necrosis, autolysis, chromatolysis, cell suicide and
several more (for review, see Majno and Joris 1995).
This partly reflects historically separate discoveries,
partly the investigators’ partiality to specific changes and
partly different forms of cell death. The most commonly
found names in today’s literature are necrosis, apoptosis
and sometimes programmed cell death.
The term ‘necrosis’, which has been used in the past
by a number of authors to describe any form of cell death,
is today – as used by most authors working on apoptosis
6
– reserved for a passive form of cell death. In this sense,
necrosis is characterised by the relatively slow disintegra-
tion of the cell without the features indicative of active
cell death (see below). The term has also been used for a
long time – and still is being used – to describe large-
scale cell death in in vivo situations when cellular rem-
nants are still visible to the examining pathologist; here it
is occasionally even used when the dead cells show clear
signs of apoptosis (Majno and Joris 1995).
The term ‘apoptosis’ is used to describe sometimes only
the morphology, sometimes the underlying mechanism, of a
cell-autonomous active process in which a specialised sig-
nalling pathway is active in killing the cell and organising
its disposal. The term ‘programmed cell death’ has for a
long time been used for developmentally occurring cell
death, when cells are ‘programmed’ to die during normal
development. It is now sometimes used as a synonym for
apoptosis (on the basis that the death of a cell by apoptosis
follows an intracellular ‘program’). Recently, attempts have
been made to revise the terminology (Samali et al. 1999),
but this incongruous use is likely to continue for some time.
The morphology of apoptotic cell death will be dis-
cussed below. In particular, this encompasses the following
aspects: What defines apoptosis and how are the morpho-
logical changes initiated? What alterations can we observe
and what do we know about their molecular origin? What
is the role and what are the consequences of these changes
for apoptotic cell death in a multicellular organism?
How uniform are the changes in apoptotic
cell death?
Cells dying by apoptosis in most situations display a
very similar pattern of morphological changes. This uni-
formity has contributed much to the emergence of the
understanding that apoptotic cell death in probably all
physiological situations is implemented by the same in-
tracellular pathway. Regardless of which situation is in-
vestigated or which agent is used to induce apoptosis ex-
perimentally, the appearance of cell death at least has
some similar features, which are often exactly the same.
However, exceptions have been reported and this uni-
form view may to some degree be an oversimplification.
Undoubtedly, morphological (and biochemical) differ-
ences can be demonstrated in different situations of cell
death in vivo. Also, when cell death is induced experi-
mentally in vitro, differences in phenotype can be in-
duced and observed; the same cell line can display ‘full-
blown’ apoptosis when treated with one stimulus and can
show only a part of the morphological changes when
treated with another, apparently indicating that cell death
is initiated by either different cell death systems or by
only part of the apoptotic pathway. It must therefore be
acknowledged that differences do exist in the develop-
ment of morphological changes even when it can hardly
be doubted that apoptosis is the underlying mechanism.
There are, however, several considerations which should
be taken into account and which may explain the varying
outcome of a process which is basically the same.
First, in many cases the observation can only be incom-
plete – for example, in in vivo situations where perhaps
only a part of the morphology can be seen before the cells
are either taken up by neighbouring cells or start to disin-
tegrate. Second, in experimental situations in vitro, the
choice of the apoptosis-inducing agent may affect the
morphological outcome; for example, when the promiscu-
ous kinase-inhibitor staurosporine is used to induce apop-
tosis it will initiate the classical apoptotic pathway but will
at the same time inhibit a large number of cellular kinases
whose action in turn may be required for normal apopto-
sis; although staurosporine-induced cell death has most of
the classical features of apoptosis, the appearance of apo-
ptotic bodies (see below) does not take place (Cotter et al.
1992). Third, cell types will differ in the expression of
proteins which determine part of the morphological result.
Such differences have prompted investigators to sug-
gest additional terms such as ‘oncosis’ for a situation,
mainly in osteocytes, where swelling of the dying cell is
a prominent feature (Majno and Joris 1995). During
metamorphosis of insects, cell death has been observed
which appears morphologically distinct – at least in
some respects – from apoptosis (Bowen et al. 1996).
When apoptotic cell death during normal development is
genetically impossible, some cell death still occurs but
without the common features of apoptosis (Chautan et al.
1999). The same stimulus can occasionally induce both
apoptotic changes and another type of morphology nor-
mally described as necrosis (Russell et al. 1972), espe-
cially if the apoptotic signalling pathway is experimen-
tally inhibited (Vercammen et al. 1998).
Not enough is known about these forms of cell death,
or about their molecular cause or natural occurrence, to
be able to discuss them as being different from apopto-
sis. It will suffice to acknowledge the possibility that not
all morphological features of apoptosis may be present
and other forms of cell death may exist. The discussion
below will focus on what we believe to be the events
during typical apoptosis.
Morphological changes occur consecutively
to apoptotic signalling events
Many authors distinguish a series of stages of apoptosis.
It has indeed been useful to separate occurrences of apop-
tosis which serve the initiation of the intracellular path-
way from events which are critical for the implementa-
tion of the death of the cell and those which are, although
very useful as indicators of apoptosis, actually dispens-
able for cell death. The most notable example of the latter
type of event is the nuclear changes during apoptosis:
these changes are very prominent and impressive to the
observer, but the death of the cell will occur just the same
in the absence of a nucleus (Jacobson et al. 1994;
Schulze-Osthoff et al. 1994). Such changes, which are
not essential for the death of the cell, are often classified
as ‘postmortem’ events, although it must be said that the
best approximation of the time of the death of a cell is
still the definition of a ‘point of no return’. ‘Postmortem’

events – and probably all morphological changes could
be classified as such – are perhaps largely dispensable for
the actual death of the cell, i.e. the cessation of the coor-
dinated cellular metabolic activities. They do, however,
probably serve an important purpose, that is the organisa-
tion of disassembly and disposal of the apoptotic cell.
The biochemical initiation of morphological changes
More than 30 years ago it was noted that apoptotic cell
death – at least in some cases – can require RNA and
protein synthesis and can thus be considered an active
process (Tata 1966). Almost 30 years ago, the observa-
tion that apoptotic cell death under very different cir-
cumstances has an almost identical appearance has
raised speculation that all apoptosis is the consequence
of the same sequence of intracellular events (Kerr et al.
1972). The molecular definition of apoptosis-associated
requirements has by now furnished us with a clearer un-
derstanding of this situation. The recognition that the
pathway to developmental cell death in the nematode
Caenorhabditis elegans is fundamentally regulated by
the same principles as apoptotic cell death in humans has
greatly aided the process of understanding cell death
(Ellis et al. 1991b).
The ced-3 gene is essential for developmental cell
death in C. elegans. The discovery that Ced-3 is a cys-
teine protease, together with the cloning and functional
characterisation of a number of Ced-3 homologues in
mammals, has had enormous impact in the science of ap-
optosis. These proteins, now called caspases, are recogni-
sed to form a common class in evolutionarily widely sep-
arated organisms. Although the death of a cell can occur
in the absence of caspase activity (for review, see Borner
and Monney 1999), the sum of available data suggests
that apoptotic cell death requires the presence of casp-
ases. It is conceivable that in some situations in vivo cells
die by other means than as a consequence of caspase acti-
vation but there can be little doubt that caspases are re-
quired for at least the more prominent morphological
changes during apoptosis (see below for details).
Caspases are catabolic enzymes whose role it is to di-
gest other proteins, and a number of structural proteins
7
can be such caspase substrates. This constellation has
initially given rise to the speculation that apoptosis is the
literal collapse of a cell whose skeleton has broken down
(Martin and Green 1995a). There are, however, now ex-
perimental results which argue that not the immediate
caspase-mediated destruction but the action of mecha-
nisms further downstream, i.e. components which be-
come activated by caspases, account for at least the
greater part of the apoptotic phenotype.
Observation of apoptosis: summary
of the prominent changes in morphology
In vivo, i.e. in animals, cell death can occur either in indi-
vidual cells scattered in tissues (which is probably more
often the case during physiological cell death) or in big-
ger, continuous areas (such as following an injury). Apo-
ptotic cells are rapidly engulfed and digested by neigh-
bouring cells (Ellis et al. 1991b; Kerr et al. 1972). This
makes it difficult to follow morphological changes in vi-
vo. However, a large body of evidence suggests that
changes induced by various agents or insults to a cell in
vitro reflect the physiological events in vivo, and the ma-
jority of studies have thus focussed on the investigation
of either regulation or morphology of apoptosis in vitro.
Apoptosis can often easily be detected under the micro-
scope. Some changes can be seen by light microscopy,
sometimes with the aid of specific dyes, and some chang-
es can be detected only by electron microscopy. The fol-
lowing morphological changes can be observed (Fig. 1):
Fig. 1 Schematic representation of the morphological changes
during apoptosis. A cell dying by apoptosis at various stages, pro-
ceeding from left. Early changes involve condensation of nuclear
chromatin along the perimeter of the nucleus and, dependent on
the context, separation from the surrounding cells in the tissue and
from extracellular matrix attachments. Blebs appear (arrows), and
the nucleus condenses completely and segregates into several
fragments. Organelles are generally intact although dilatation of
the ER and release and aggregation of ribosomes have been ob-
served. Sometimes cytoplasmic vacuoles can be seen. The cell dis-
integrates into apoptotic bodies which can contain any part of the
cellular material. In the final step, apoptotic bodies are taken up by
other cells and digested via a lysosomal pathway
8
As observed by light microscopy
The dying cell starts to sever attachments to other cells
and extracellular matrix and to round up (a feature obvi-
ously most easily observed in adherent cells, less easily
visible in cells in suspension culture). It starts to show
protrusions from the plasma membrane, commonly re-
ferred to as ‘blebs’. Blebs can be observed for some time
protruding and retracting, especially if later processes
are inhibited experimentally. DNA staining with certain
dyes allows the direct observation of the condensation of
the cell nucleus, which often starts as a condensed
ring/sphere along the nuclear envelope which will extend
to encompass the entire nucleus. The condensed nucleus
can be seen to disassemble into several fragments. The
entire cell condenses and is reorganised into so-called
‘apoptotic bodies’, a term coined by Kerr et al. (1972).
Apoptotic bodies are membrane-bound vesicles which
vary in size and composition; they can contain the entire
contents of the cell in various combinations, such as cy-
tosolic elements, organelles or parts of condensed nuclei
(Fig. 1).
As observed by electron microscopy
In addition to the changes seen by light microscopy,
changes to organelles have been described by electron
microscopy. Most studies agree that mitochondria re-
main intact for at least some time. Then they have been
described to either condense or to swell (perhaps de-
pending on the cellular context). Intracellular membrane
compartments such as the endoplasmic reticulum and
Golgi apparatus also appear to be morphologically nor-
mal at least during the early stages of apoptosis and
probably become involved only in the late stages; how-
ever, dilatation of the ER has also been noted earlier on.
Vacuolation of the cytoplasm has also been described in
various studies. Electron microscopy can also reveal the
loss of microvilli during apoptosis.
Table 1 Summary of common morphological findings and their known or likely molecular origin. The indicated molecular mechanism
is partly speculative. Caspase activity means that caspases are involved but the precise mechanism is unknown. Refer to text for details
Cellular target Phenotypic change
Nucleus Condensation, disintegration
Cell shape and structure Detachment, blebbing,
disassembly into apoptotic
bodies
Mitochondria Swelling(?), condensation(?)
Endoplasmic reticulum Dilatation
Cytosol Vacuolisation
Plasma membrane Loss of microvilli
Fig. 2 Effectors of morphological changes during apoptosis.
Caspases (mainly caspase-3, probably caspase-7 and caspase-6)
are activated and their activity is required for the observed chang-
es in morphology. Components of focal adhesions such as focal
adhesion kinase (FAK) are cleaved and perhaps mediate detach-
ment of the cell. DFF and Acinus are direct caspase substrates
which cause nuclear condensation and probably fragmentation by
a not entirely clear mechanism. Direct caspase cleavage of further
nuclear substrates may also be involved. Rabaptin5 is cleaved by
caspases during apoptosis. Its role in the appearance of cytosolic
vacuoles and its effect on changes in the intracellular membrane
systems is largely speculative. Blebbing and the appearance of
apoptotic bodies requires caspase activity but the molecular link is
somewhat unclear. Actin reorganisation (at one stage probably in-
to a ring or a sphere along the plasma membrane) appears to play
a role in this process. Myosin II phosphorylation (by myosin light
chain kinase, MLCK) and the action of further myosins could be
the driving force of the extrusion and retraction of the blebs; it has
been proposed that points of local weakness allow this protrusion
(Mills et al. 1999). Kinases such as p21-activated kinase 2 (Pak2)
and Ste20-related kinase (SLK) are likely to be involved in actin
restructuring, perhaps following direct proteolytic activation by
caspases. Dotted lines show speculative and perhaps indirect links.
Only some of the proposed mechanisms are included. See text for
further details
Molecules involved Initiating mechanism
DFF, Acinus lamins(?), Cleavage by caspase-3,
scaffold attachment factor(?) caspase-dependent cleavage(?)
Focal adhesion kinase, Hsp27, Caspase activity, caspase-dependent
Pak2, SLK, myosin II, cleavage of Pak2 and SLK,
other myosins(?), caspase activity, phosphorylation(?),
actin reorganisation, fodrin(?), kinase activity(?), caspase-dependent
gelsolin(?), F-actin, Pak2 cleavage, restructuring,
caspase-dependent cleavage,
caspase activity
? ?
Rabaptin5(?) Cleavage by caspases(?)
Rabaptin5(?) Cleavage by caspases(?)
Ezrin/radixin/moesin proteins Dephosphorylation(?)

At some stages, apoptosis will affect probably all the
components and organelles in a dying cell. Some of
these changes are direct consequences of the action of
the apoptotic signalling pathway and should therefore be
considered apoptotic changes in the strict sense. At later
stages – if the apoptotic cell is not taken up and degraded
by another cell – changes will take place which at least
partly originate from the loss of a coordinated metabolic
activity and compartmentalisation. Such changes should
perhaps be best referred to as secondary.
In the following sections, the molecular context of the
individual morphological changes will be discussed. A
summary of the likely and more speculative molecular
mechanisms is given in Fig. 2 and Table 1.
Molecular basis of the morphological alterations
to the individual elements of a cell
Nuclear changes
Nuclear changes play a predominant role in the many de-
scriptions of apoptosis. This prevalence is, however, not
so much the expression of their factual importance for
the apoptotic process as of the relative ease of experi-
mental detection. Indeed, apoptotic cell death in proba-
bly all other respects can proceed normally in cells
which carry mutations prohibiting normal nuclear apop-
tosis (Ucker et al. 1992; Kawabata et al. 1999) or even in
enucleated cells (Jacobson et al. 1994; Schulze-Osthoff
et al. 1994). The detectable morphological changes in the
nucleus are chromatin condensation and, at a later stage,
the fragmentation of the nucleus into several particles
(Fig. 1). With the disintegration of the cell, these parti-
cles are packaged into apoptotic bodies and taken up by
phagocytes and neighbouring cells (Kerr et al. 1972).
The changes in nuclear appearance can be detected by
electron microscopy or also very clearly by light micros-
copy with DNA-intercalating dyes such as acridine or-
ange or Hoechst dyes to visualise the nucleus.
The earliest detectable alteration is the condensation
of the nuclear chromatin into more densely packed mate-
rial along the nuclear membrane. This is discernible as
electron-dense matter under the electron microscope and
results in a ring with a brighter stain when fluorescent
dyes are used. The condensation then often encompasses
the whole nucleus, and the condensed nucleus starts to
disintegrate into similarly dense, smaller particles which
are packaged and taken up. A number of specific apopto-
sis-associated biochemical events have been defined to
occur around the same time and have been suggested to
participate in the implementation of the morphological
changes: caspase-mediated proteolysis of nuclear pro-
teins and degradation of the chromosomal DNA. Recent
studies have also proposed that specialised proteins act
as separate effector molecules in this process (see be-
low).
Active caspases, probably predominantly caspases-3
and -6, cleave a number of nuclear proteins such as DFF
9
45 (ICAD), PARP, lamins, nuclear scaffold attachment
factor and others (Liu et al. 1997; Lazebnik et al. 1994,
1995a; Gohring et al. 1997; Hirata et al. 1998). How
caspases – which normally reside in the cytosol – gain
access to nuclear proteins is not clear but it would appear
likely that they need to be present in the nucleus to
cleave their substrates. Caspase-2 (which has an uncer-
tain role in apoptosis) has been found in the nucleus
even in resting cells (Colussi et al. 1998), and caspase-3
has been reported to be translocated to the nucleus dur-
ing apoptosis (Nakagawara et al. 1997; Zhivotovsky et
al. 1999). The mechanism of such possible translocation
is unclear.
Although this cleavage is undoubtedly achieved by
caspases during apoptosis, the currently available data
suggest that at least part of these cleavage events may be
fortuitous or at least – for some situations investigated –
dispensable for the normal occurrence of apoptosis.
Nuclear lamins were among the earliest proteins to be
discovered as caspase substrates and have perhaps for
this reason received more attention than ‘later’ sub-
strates. Cleavage of lamins in mammals as well as in
Drosophila during apoptosis has been reported (Lazebnik
et al. 1995a, 1995b; Fraser et al. 1997). Lamins are nu-
clear proteins which are attached to the inner side of the
nuclear envelope and whose function is not entirely
clear, although a number of roles have been proposed for
them (Moir and Goldman 1993). Whether the cleavage
of nuclear lamins contributes to the nuclear breakdown,
i.e. the observed chromatin condensation, is debatable.
Studies expressing uncleavable mutants of either lamin
A or lamin B have found that such overexpression can in
both cases prevent or at least delay changes in nuclear
morphology (Rao et al. 1996). These results would sug-
gest that the cleavage of both proteins is required, i.e.
that both lamins exert different functions in preserving
nuclear integrity which could fill in for each other. Re-
sults from a perhaps somewhat rough approach from our
laboratory argue that lamin cleavage does not contribute
appreciably to the appearance of nuclear morphological
changes: when cells were loaded with proteinase-K, at
least lamin B was completely digested (and very likely
other lamins as well) but this was not sufficient to cause
nuclear condensation; when caspases were active at the
same time, typical nuclear apoptotic changes were seen
(Wilhelm and Häcker 1999). It is therefore conceivable
that nuclear morphological changes do not require the di-
gestion of nuclear structural proteins by caspases – this
perhaps facilitates and emphasises changes – but that
these changes are the result of the activation of ‘speciali-
sed’ caspase substrates, of molecules which have no oth-
er function than implementing the death signal transmit-
ted by caspases. It may also be that caspase-mediated
cleavage is dispensable for the early condensation but re-
quired for the final separation of the nucleus into smaller
bodies.
The biochemically prominent feature of nuclear apop-
tosis is the degradation of the chromosomal DNA and
the possibility has to be considered that this event is
10
causally linked to the change in morphology. Chromosom-
al digestion results first in large (30–50 and 200–300 kbp)
DNA fragments; then, internucleosomal cleavage results
in the appearance of fragments of multiples of the length
of DNA around the nucleosome (Wyllie 1980); both pat-
terns are likely to be linked (Enari et al. 1998; Sakahira
et al. 1998). This sign of apoptosis can be observed easi-
ly by extraction and agarose-gel electrophoresis, which
will yield a ladder-like pattern of DNA (Wyllie 1980).
Enzymatic labelling of free DNA ends allowing the spe-
cific detection of DNA-strand breaks [so-called TUNEL
(TDT-mediated dUTP-biotin nick end-labeling) reaction;
Gavrieli et al. 1992] is also commonly used. By these
methods, it is possible to investigate DNA fragmentation
and nuclear morphological changes in parallel.
The observation that in most of the observed situa-
tions the two events of DNA degradation and chromatin
condensation have occurred together might mean that
they depend on the same molecular events and are
caused by the same effector mechanism. Recently report-
ed results now suggest that the two might be separate en-
tities.
It has been the objective of a number of studies over
the past 2 decades to discover the nature of apoptotic
chromatin condensation and DNA degradation and the
link between the two, both in intact cells and using iso-
lated nuclei (for example, see Cohen and Duke 1984; To-
mei et al. 1993). Varying results have been obtained:
simple digestion of the chromosomal DNA with micro-
coccal nuclease has been found to induce chromatin con-
densation in one (Arends et al. 1990) but not another
study (Sun et al. 1994). When endogenous nucleases
were activated with divalent cations, both events ensued;
in this system, DNA degradation could then be inhibited
by Zn ions while chromatin condensation proceeded un-
impeded (Sun et al. 1994). Valuable as such studies are,
the obvious problem is that the events triggered by such
agents may be different from the mechanisms operative
during apoptosis. In this respect, the cloning and charac-
terisation of two molecules named DNA-fragmenting
factor (DFF) 45 and 40 (in humans) or ICAD/CAD (in
mice) about 2 years ago have significantly contributed to
our knowledge of nuclear destruction during apoptosis.
DFF/CAD/ICAD appears to be normally synthesised
in many or all tissues and assumes the form of an inac-
tive dimer, in which the active component DFF40 or
CAD is complexed and kept inactive by the inhibitor and
chaperone DFF45 or ICAD (Liu et al. 1997, 1998; Enari
et al. 1998; Sakahira et al. 1998). Although two groups
have independently purified the proteins from cytosolic
extracts (Liu et al. 1997; Enari et al. 1998), the normal
localisation of DFF is likely to be nuclear (Liu et al.
1998; Samejima and Earnshaw 1998a). During apopto-
sis, DFF45 is cleaved by activated caspases (probably
mainly caspase-3 but possibly also others) (Janicke et al.
1998a, 1998b; Tang and Kidd 1998), and releases
DFF40. DFF40 causes the typical double-stranded DNA
breaks – when nuclei are used as targets, they first occur
at the more accessible sites between nucleosomes as is
the case in vivo. Possibly, this degradation is achieved in
concert with other nuclear proteins such as histones and
HMG proteins and perhaps after oligomerisation of
DFF40 (Toh et al. 1998; Liu et al. 1999).
Conflicting results and suggestions can be found in
the literature regarding the question of whether chroma-
tin condensation is a necessary consequence of DFF40
activation. Genetically modified mice without functional
CAD showed defects in both DNA degradation and
chromatin degradation (Zhang et al. 1998). The treat-
ment of cell extracts with recombinant caspases led to
chromatin condensation in cell-free systems, and this ac-
tivation could be blocked by recombinant, uncleavable
DFF45 (Wöhrl and Häcker 1999), and purified DFF40
was found capable of inducing chromatin condensation
(Liu et al. 1998); both results argue for a role of DFF in
nuclear condensation. In a cell line, however, which ex-
pressed an uncleavable DFF variant, DNA degradation
was prevented but chromatin condensation still occurred
(Sakahira et al. 1999). Furthermore, experiments using
extracts from apoptotic cells suggested that an activity
other than DFF was required for chromatin condensation
(Samejima et al. 1998b). A molecule has been cloned re-
cently and named Acinus which appears to fulfil these
criteria: it can be activated by caspases and induce only
chromatin condensation but not DNA fragmentation
(Sahara et al. 1999). Perhaps differences in the types of
cells used for these studies could account for the varia-
tions, or maybe the experimental situations employed
have provoked biochemical events not seen normally
during apoptosis. It is too early to derive a complete pic-
ture of the physiology of nuclear destruction during ap-
optosis but the discoveries of the past few years have
greatly furthered our understanding of this aspect of the
biology.
Following condensation, the nucleus is broken up and
separates into a number of fragments which are consecu-
tively packaged into apoptotic bodies. The molecular
events responsible for this are unknown but caspase-me-
diated cleavage of nuclear proteins may be involved.
Another protein termed AIF has been suggested to act
as an effector molecule in nuclear apoptosis. AIF nor-
mally resides in mitochondria and can be released by
apoptotic stimuli. AIF can induce some morphological
alterations in nuclei, in particular chromatin condensa-
tion in the outer layers of the nucleus but probably not
complete condensation and fragmentation (or internu-
cleosomal degradation of DNA) (Susin et al. 1996,
1999). The physiological importance of this molecule for
apoptosis and for nuclear morphological changes is un-
clear.
In conclusion, we should ask about the relevance of
nuclear apoptosis for the organism. As far as we can tell,
the nuclear events of apoptosis are not essential for the
death of the cell; some morphological changes of apop-
tosis have even been demonstrated in the absence of a
nucleus (see above). On the other hand, with DFF and
perhaps Acinus, a system exists which seems specialised
only in the destruction of the nucleus following caspase

activation and has, it would appear, afforded sufficient
advantage during evolution to permit selection. An endo-
nuclease in C. elegans participates in the normal disposal
of the cells which have died by programmed cell death
(Ellis et al. 1991a); perhaps the machinery in higher or-
ganisms serves a similar purpose, i.e. to degrade nuclear
waste in order to facilitate the disposal of the dead cell
by phagocytes. It is also conceivable that DNAse activa-
tion is useful in the digestion of dangerous, particularly
viral, DNA and that this mechanism functions primarily
to exclude the possibility of transmission of viral genetic
information from a dead cell to a phagocytosing cell.
Changes to shape and structure of the cell undergoing
apoptosis
One of the most striking changes during the observation
of apoptosis under the microscope is the reorganisation
of the outer limits of the cell and the changes in shape.
Cells undergoing apoptosis round up and sever contacts
to neighbouring cells in tissues. Protrusions from the cell
surface become visible both by light and electron mi-
croscopy. These protrusions, the so-called ‘blebs’, have
at one stage prompted researchers to coin the name ‘pop-
corn cytolysis’ (Russell et al. 1972). Blebbing continues
for varying times – if the downstream events are inhibit-
ed experimentally, for about 1 h (Mills et al. 1999); in
what appears to be a separate stage, the cell condenses –
hence the once common term ‘shrinkage necrosis’ – and
disintegrates into ‘apoptotic bodies’, i.e. several mem-
brane-bound vesicles which contain cellular organelles,
cytoplasm and nuclear fragments (Kerr et al. 1972). Vac-
uoles have been observed to appear in the cytoplasm of a
dying cell. A number of aspects of this process have also
been discussed in a recent review article (Mills et al.
1999).
The basis of these changes is probably the concerted
reorganisation of cytoskeletal structures.
It is not entirely clear whether the detectable altera-
tions are consecutive events caused by one pathway and
possibly depend on each other or whether they are regu-
lated separately. It can be assumed that caspases play a
role in the appearance of these alterations under normal
circumstances, although, in some experimental systems,
the chemical inhibition of caspases had only partially in-
hibitory effects (Mills et al. 1998b). The clearest result
concerning the question of such a contribution comes
from studies with mice lacking caspase-3. In these ani-
mals, at least hepatocytes and thymocytes (no other cell
type was investigated) showed a strong reduction in
blebbing (Zheng et al. 1998). Similarly, a human breast
cancer cell line lacking caspase-3 was found to be defec-
tive in blebbing during cell death (Janicke et al. 1998b).
This implies that structural events are indeed the conse-
quence of caspase activation.
How caspases achieve the changes is less clear. A
number of structural proteins is cleaved by caspases di-
rectly during apoptosis, for instance gelsolin, fodrin, ac-
11
tin or Gas2 (Kothakota et al. 1997; Martin et al. 1995b;
Mashima et al. 1995; Brancolini et al. 1995). For most of
these caspase targets the actual contribution to cytoskele-
tal rearrangement is at least not obvious. As already re-
ferred to, loading of cells with proteinase-K leads to the
digestion of intracellular proteins. Such loading also in-
duces blebbing and the formation of apoptotic bodies. In
the presence of caspase inhibitors, this proteinase-K-in-
duced blebbing is abolished although cellular proteins
are still degraded (Wilhelm and Häcker 1999). This indi-
cates that – if direct caspase cleavage of cytoskeletal
proteins plays a role in this process – it is not the mere
degradation but proteolytic activation of downstream tar-
gets which will result in morphological changes.
Caspase-mediated cleavage of proteins which partici-
pate in the organisation of focal adhesions (such as focal
adhesion kinase, FAK) may contribute to the early de-
tachment of cells from surrounding matrix (Wen et al.
1997; Levkau et al. 1998; Bannerman et al. 1998); mi-
crotubule disassembly might also play a role here (Mills
et al. 1998a). Studies with the inhibitor cytochalasin D
suggest that actin reorganisation is required for the for-
mation of blebs and such reorganisation of actin into a
ring-like structure along the cell periphery has been de-
scribed (Huot et al. 1998). The protein gelsolin, which is
involved in the organisation of the actin network, has
also been implicated in apoptosis. Besides a possible role
in the initiation of apoptosis – overexpression of gelsolin
has been shown to make cells more resistant to various
apoptotic stimuli (Ohtsu et al. 1997) – this molecule has
also attracted attention as a target of caspases during ap-
optosis. A caspase-generated fragment of gelsolin can
disassemble actin filaments in vitro, and cells from gels-
olin-k.o. mice show a delay – but only a delay – in
blebbing (Kothakota et al. 1997). The same cells, how-
ever, also display a delay in DNA degradation. This is
not readily comprehensible if gelsolin is a direct effector
downstream of caspases on the way to structural changes
and suggests that indirect effects are involved as well.
The process of blebbing has recently been investigat-
ed in several careful studies. Although blebbing appears
to require for its initiation caspase-3 activity, once start-
ed, it can continue in the presence of caspase inhibitors
(which at the same time block the formation of apoptotic
bodies, suggesting that they are indeed effective in inhib-
iting caspases) (McCarthy et al. 1997; Mills et al.
1998b). This again indicates that caspases activate down-
stream mechanisms which mediate the appearance of
blebs. The actual blebbing has been found to depend to
some extent on actin polymerisation (Mills et al. 1998b).
Studies both on non-apoptotic (suprastimulation of pan-
creatic acinar cells; Torgerson and McNiven 1998) and
on apoptotic blebbing (Mills et al. 1998b) have attributed
an active role to myosin in the protrusion of the blebs.
This myosin action was shown to depend on its phos-
phorylation [mainly by myosin light chain kinase
(MLCK), possibly to a smaller extent following activa-
tion of members of the Rho family of small G proteins
(Mills et al. 1998b)]. A model has been put forward pro-
12
posing that apoptotic blebs are protrusions at a focal
point of weakness in a contracted actin ring (Mills et al.
1999). Taken together, these data suggest that one prod-
uct of caspase cleavage leads to the activation of myosin
light chain kinase and that a form of actin-myosin inter-
action participates in the protrusion and retraction of
apoptotic blebs. The molecular link between caspase ac-
tivity and kinase activation, however, is unclear.
Reorganisation of the cytoskeleton can normally be
mediated by the activity of a number of kinases and
some of them have been implicated in the morphological
changes during apoptosis. Mitogen-activated protein
(MAP) kinase-dependent phosphorylation of heat shock
protein 27 has been suggested to be involved in the ac-
tin-restructuring of blebbing (Huot et al. 1998). More di-
rectly, the p21-activated kinase 2 (Pak2) has been shown
to be activated by caspase-mediated cleavage (Rudel and
Bokoch 1997; Lee et al. 1997). Pak2 can participate in
remodelling of the actin-cytoskeleton and could thus be
involved in the apoptotic restructuring. Since an un-
cleavable mutant of Pak2 also delays apoptosis, it is,
however, uncertain whether the role of Pak2 cleavage is
limited to downstream morphological events of apopto-
sis, and the observed effects could at least partly be indi-
rect (Lee et al. 1997).
Recent work has also implicated the novel Ste20-re-
lated kinase, SLK, in a similar way in actin rearrange-
ment during apoptosis. SLK is cleaved by caspase-3 dur-
ing apoptosis and the fragments were shown to be able to
rearrange cytoskeletal components (Sabourin et al.
2000). Perhaps such kinase activation is one principle of
how blebbing is accomplished.
The molecular events of the final shrinking and the
formation of apoptotic bodies are relatively poorly un-
derstood. Pak2 may be involved in the formation of apo-
ptotic bodies (Rudel and Bokoch 1997) and, since this
process is inhibited by cytochalasin B, a participation of
F-actin is possible (Cotter et al. 1992). It has, in other
studies, been suggested that caspase activity is required
for the formation of apoptotic bodies independently, i.e.
downstream, of bleb formation (McCarthy et al. 1997;
Mills et al. 1998b). This argues against a direct contribu-
tion from Pak2 (at least if the role of Pak2 in blebbing is
an essential one) and calls for identification of the casp-
ase substrate or substrates involved. No candidate has
been discovered so far.
The appearance of cytosolic vacuoles can further be
seen during apoptosis (Kerr et al. 1972). Although this
observation has been made numerous times, the underly-
ing molecular mechanism is unclear (Henics and Wheat-
ley 1999). In one study it has been found that the vacuo-
lation required caspase activity (Fearnhead et al. 1995)
while other work has described the appearance of vacu-
oles despite caspase inhibition (Xiang et al. 1996). One
can speculate that the caspase-mediated cleavage of
Rabaptin5 – which was proposed to disturb membrane
turnover in an apoptotic cell (see below) – is involved.
In summary, a large number of effectors have been
implicated in the changes of cell shape and structure dur-
ing apoptosis. It is worth noting that all of them are not
specific for apoptosis but are effector proteins which can
induce similar changes also in normal, non-apoptotic
cells. While it is certainly possible that these effector
molecules are indeed used by the apoptotic pathway,
from today’s point of view this would be a difference
from nuclear disassembly, where specialised proteins
such as DFF and perhaps Acinus and CIDE (Inohara et
al. 1998) appear to exist. This again might mean either
that such molecules – specialised in apoptotic reshaping
– are yet to be discovered or, if they do not exist, that the
structural changes have more the characteristics of fortu-
itous occurrences and less the characteristics of essential
contributing events of apoptosis.
Changes to mitochondria
The initial ultrastructural studies of apoptosis agree that
shape and structure of mitochondria remain intact struc-
turally until late in the process of apoptosis (Kerr et al.
1972; Belloni et al. 1982; Bardon et al. 1987) and still
maintain at least a degree of metabolism for a long time
(Pipan and Sterle 1979). After these early studies, atten-
tion has focussed on mitochondria in waves: first, when
the antiapoptotic protein bcl-2 was presumed to be locat-
ed in the inner mitochondrial membrane (this is now be-
lieved not to be the case); second, when an activity was
demonstrated in mitochondria which was reported to in-
duce nuclear apoptotic changes upon release (Susin et al.
1996); and, third, when the mitochondrial protein cyto-
chrome c was shown to be able to activate caspases (Li
et al. 1997). The focus of these studies has been the
functional aspect in this relationship, i.e. the possible
role of mitochondria in the induction of apoptosis; how-
ever, some morphological considerations can be deduced
as well.
A great number of studies found molecular changes in
mitochondria at an early time point, more or less around
the same time as the activation of caspases. The ob-
served changes are largely not morphological but consist
of invisible molecular changes to mitochondria, especial-
ly the release of mitochondrial proteins and changes in
ion gradients and electrochemical gradients (for review,
see Green and Reed 1998). On the morphological level,
mitochondria have been described as either unchanged
or even condensed during apoptosis (Kerr et al. 1972;
Sheridan et al. 1981; Russell et al. 1972). Because cyto-
chrome c, which is normally contained within the mito-
chondrial membranes, can upon release activate casp-
ases, the possibility of a physical disruption of the outer
mitochondrial membrane as a structural indication has
been investigated. Indeed, in contrast to earlier studies,
one report describes a swelling of mitochondria leading
to the rupture of the outer mitochondrial membrane de-
tectable by electron microscopy and the release of cyto-
chrome c at an early stage of apoptosis (Vander Heiden
et al. 1997). Recently reported work, on the other hand,
found that the addition of the proapoptotic proteins Bid

or Bax led to the release of cytochrome c and other pro-
teins from the intermembrane space of isolated mito-
chondria in the absence of swelling (Kluck et al. 1999).
This would suggest that mitochondria can at least in the
early stages of apoptosis remain physically intact; in the
later stages, a condensation is possible.
Changes to further organelles and degradation
of apoptotic bodies
Reports are sparse on the changes to further organelles.
Kerr and others noted in the late 1960s that organelles –
mitochondria, endoplasmic reticulum (ER), lysosomes –
appear to be intact in the apoptotic process, at least until
most of the other morphological changes are completed
(Kerr 1965; Ballard and Holt 1968; for review, see Kerr
et al. 1972). Not much new information has been added
to that.
Undoubtedly, the death of the cell will at some stage
concern all organelles. There is, however, no clear evi-
dence that morphological changes to further organelles
are directly linked to the apoptotic pathway. Dilatation of
the ER during apoptosis can be seen ultrastructurally
(Pollard et al. 1987; Cotter et al. 1992; Ludewig et al.
1995). In a study of human breast cells dying by apopto-
sis evidence was found for a detachment of ribosomes
from the rough ER as well as ribosomal aggregation
(Ferguson and Anderson 1981).
Conversely, changes to the ER or to enzymes local-
ised in the ER can cause apoptosis (Zinszner et al. 1998;
Qi et al. 1997). The integral ER-protein Bap31 has been
demonstrated to bind both Bcl-2 and pro-caspase-9 in the
ER (Ng et al. 1997; Ng and Shore 1998). Cleavage of
Bap31 during apoptosis by caspases (Granville et al.
1998) has been demonstrated. A causal connection be-
tween these biochemical apoptotic events and the ob-
served dilatation can, however, be only speculative.
It has been described that caspase-mediated cleavage
of Rabaptin5 can prohibit endosomal membrane fusion
(Cosulich et al. 1997). It can be speculated that such in-
hibition will also affect other membrane systems in the
cell such as ER and the Golgi cisternae and that such dis-
turbance causes morphological changes.
Phagocytosis of apoptotic cells or of the product of
cellular disintegration, the apoptotic bodies, is without
doubt an integral feature of apoptosis. Uptake and degra-
dation of apoptotic cells in vivo is rapid and can be con-
ducted by both ‘professional phagocytes’ such as macro-
phages and non-specialised cells, for example epithelial
cells (Kerr et al. 1972). Eventually, apoptotic cells will
be digested by a lysosomal pathway. In most cases – at
least in vivo – this is probably accomplished inside the
phagocytosing cell although a link to autophagy has been
suggested. The mechanism of autophagy, i.e. the regulat-
ed, cell-autonomous autodigestion of cellular compo-
nents through a lysosomal pathway, appears to be impor-
tant for a number of cellular functions (Dunn 1994;
Liang et al. 1999). Autophagosomic vesicles have been
13
observed during apoptosis in insects (Dai and Gilbert
1999) and mammalian cells (Ohsawa et al. 1998); a pro-
tein whose expression was induced during apoptosis was
found to be a homologue of a yeast autophagy-related
protein (Hammond et al. 1998) and another mammalian
protein, Beclin, has been found to be involved in autoph-
agy and also to bind Bcl-2 (Liang et al. 1999). It is there-
fore possible that the pathway of autophagy is intercon-
nected with the pathway of apoptosis but the evidence
for this is far from conclusive.
Phagocytosed apoptotic bodies are digested by a lyso-
somal mechanism (Bursch et al. 1985). In programmed
cell death in insects, increase in lysosomal activity corre-
lated with the occurrence of cell death (Halaby et al.
1994). Inhibition of lysosomal acidification can prevent
the complete degradation of apoptotic bodies (Odaka and
Mizuochi 1999; Samms et al. 1999). The number of ly-
sosomes increased during cell death in one cell line
(Lapointe et al. 1993). It is therefore likely that uptake
by phagocytosis and degradation by lysosomal enzymes
is the end stage of apoptosis.
Changes to the plasma membrane
Changes to the plasma membrane during apoptosis are
mostly of a biochemical nature and do not seem to in-
volve morphological alterations. An exception is the loss
of microvilli which has been observed by electron mi-
croscopy in several cases and may be a common feature
of cell death (Fernandez-Segura et al. 1990; Kondo et al.
1997; Nagata 1996). Loss of microvilli has been shown
to occur downstream of caspase activation and concomi-
tantly to dephosphorylation of the ezrin/radixin/moesin
proteins, which normally stabilise microvilli to the actin
skeleton (Kondo et al. 1997).
The molecular changes in the plasma membrane are
probably important for the initiation of phagocytosis and
several candidates for ‘eat-me’ signals have been found
in various species (Ellis et al. 1991a; Rotello et al. 1994;
Franc et al. 1996; Savill 1996; Fadok and Henson 1998).
In most circumstances, phagocytosis of dead cells re-
quires the activation of the regular apoptotic pathway but
one exception has been noted: when neutrophils are pro-
tected against cell death by expression of Bcl-2, they are
still taken up and digested by macrophages (Lagasse and
Weissman 1994), suggesting that either more than one
pathway to phagocytosis exists or that the same pathway
can be triggered by different intracellular events.
Importance of morphological changes
for the organism
Striking as the apoptotic morphology is with respect to
its often clear features and its uniform appearance, the
question of its importance for the organism is a very dif-
ficult one. Undoubtedly, the mechanism of cell death it-
self is of critical importance to complex organisms but
14
are changes in morphological appearance – and the mo-
lecular events they reflect – also important? Are the de-
tectable changes purely of the ‘bystander type’; are they
only fortuitous occurrences of evolutionarily selected
signalling events? The final answer to such questions
will only be known when we have a complete under-
standing of why and to what immediate purpose apopto-
sis evolved. A number of the observed changes during
apoptosis, biochemical and morphological, are bound to
be dispensable for the concept of apoptosis in an intact
organism, as are, for example, nuclear changes for the
death of the cell in culture. A large number of caspase-
cleavage events, for instance, appear too insignificant to
warrant sufficient evolutionary pressure, at least to our
current understanding.
It is possible that the death of the cell, i.e. the cessa-
tion of the coordinated biochemical events, was the orig-
inal goal when cell death evolved; this might have been a
mechanism to combat viral infections in primitive organ-
isms (Vaux and Hacker 1995). It is also conceivable that
the recycling of dispensable cells in times of want, as has
been observed in the simple metazoan hydra, was the
driving force for the evolution of apoptosis (Cikala et al.
1999). In any case, – at least in more complex animals –
the system of large-scale cell disposal via apoptosis can
only work if an efficient removal of the dead cells is
guaranteed. Along these lines, the morphological chang-
es could well be the visible counterpart of such ‘packing-
up’ of a cell being made ready for disposal. Digestion of
chromosomal DNA and the restructuring of the cytoskel-
eton – the likely basis for membrane blebbing – could be
useful to facilitate the secondary digestion of the apo-
ptotic cell by the phagocytosing cell. Disintegration into
smaller apoptotic bodies might serve a similar purpose.
It is therefore conceivable that what we can detect as ap-
optosis though a microscope is directly linked to the evo-
lutionary goal of apoptosis.
Concluding remarks
Apoptosis research is still a fast-growing scientific disci-
pline. Although the unifying concept of cell death is gen-
erally accepted, the progress on this concept has not
been able to keep up with the accumulation of new re-
sults from literally all areas of biology. Inevitably, indi-
vidual results have been reported which appear incom-
patible with other developments, and a number of theo-
ries will have a short life (Strasser 1999). I have attempt-
ed to compile a model of induction and implementation
of morphological changes from independent reports,
from studies which pursued various lines of research.
Without any doubt, the picture is incomplete and review
articles will have to be reviewed and rewritten. Although
progress in apoptosis research has been rapid and very
considerable, it will be a number of years until we know
the complete context of both apoptotic morphology and
its underlying mechanisms.
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