Pyknosis

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Apoptosis

Pyknosis, or karyopyknosis, is the irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis [1] or apoptosis. [2] It is followed by karyorrhexis, or fragmentation of the nucleus.

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Pyknosis (from Ancient Greek πυκνός meaning "thick, closed or condensed") is also observed in the maturation of erythrocytes (a red blood cell) and the neutrophil (a type of white blood cell). The maturing metarubricyte (a stage in RBC maturation) will condense its nucleus before expelling it to become a reticulocyte. The maturing neutrophil will condense its nucleus into several connected lobes that stay in the cell until the end of its cell life.

Pyknotic nuclei are often found in the zona reticularis of the adrenal gland. They are also found in the keratinocytes of the outermost layer in parakeratinised epithelium.

Overview of Pyknosis

Pyknosis, or the irreversible nuclear condensation (a nuclear morphology) in a cell (generally old vertebrate leukocyte cells) is the result of a cell undergoing either apoptosis or necrosis. [3] There are two types of pyknosis: nucleolytic pyknosis and anucleolytic pyknosis. Nucleolytic pyknosis occurs during apoptosis (a form of controlled/programmed cell death), while anucleolytic pyknosis occurs during necrosis. [4] Necrosis is a form of regulated cell death due to toxins, infections, and other acute stressors. [4] These stressors cause swelling/shape modification of cellular organelles leading to the eventual loss of stability and integrity of the cell membrane. [4]

In simpler terms, pyknosis is the process of nuclear shrinkage that may occur during both necrosis and apoptosis. Pyknosis is also characterized by eventual fragmentation (karyorrhexis) of the dense nuclear chromatin, resulting in dark, round, and dense nuclear fragments. [5] Karyorrhexis is the fragmentation of a pyknotic cell’s nucleus and the cleavage and condensing of chromatin. [5]

Pyknosis provides a distinction between apoptosis and necrosis

Morphological characteristics of pyknosis and other forms of nuclear destruction Nuclear changes.jpg
Morphological characteristics of pyknosis and other forms of nuclear destruction

Apoptosis is characterized by nuclear condensation, shrinking of the cell, and blebbing of the nuclear and cell membrane, while necrosis is characterized by nuclear condensation, swelling of the cell, and breaks in the cell membrane. [6] Both necrosis and apoptosis are regulated by a few of the same proteins: caspase-activated DNase (CAD), endonuclease G and DNase I. Pyknosis occurs in both an apoptotic and a necrotic cell. Pyknosis in an apoptotic cell is identified by nuclear condensation, chromatin fragmentation, and the formation of a few large clumps which are enveloped by apoptotic extracellular vesicles, which are to be released when the cell dies. [6] Pyknosis in a necrotic cell is identified by nuclear condensation and fragmentation into small clumps that will be dissolved later in the process of the necrotic cell’s death. [6] Consequently, pyknosis can be distinguished into two types, nucleolytic pyknosis (apoptotic cells) and anucleolytic pyknosis (necrotic cells).

The types of pyknosis

Nucleolytic pyknosis

Nucleolytic pyknosis, which can also be referred to as apoptotic pyknosis, involves three main events. These are disrupting the nuclear membrane, the condensing of the chromatin, and lastly, nuclear cleavage/fragmentation. [4] Throughout these events the cell shrinks in size and the cell membrane undergoes blebbing, which is the forming of membrane bulges across the exterior-facing surface of the cell membrane. During the first event (the disruption of the nuclear membrane), several enzymes are used to cleave the proteins found in the nuclear membrane. These enzymes, caspase-3 and caspase-6, both target and cleave nuclear membrane proteins, including NUP153, LAP2, and B1 (proteins that are used for membrane structure and molecular transport). [4] This cleavage, in turn, results in a disruption of the interior of the membrane, which is an initiating factor for chromatin condensation (the second event of nucleolytic pyknosis). This is due to the fact that caspase-3 cleaves Acinus, which has DNA/RNA binding domains and ATPase activity to initiate the condensation of chromatin. [4]

Anucleolytic pyknosis

Anucleolytic pyknosis, which can also be referred to as necrotic pyknosis, involves the swelling of the cell, followed by the separation of the nuclear membrane from chromatin, the eventual collapse of both the nuclear membrane and chromatin, and finally the cell membrane ruptures (the cell dies). [4] One protein that plays a significant role in necrotic pyknosis is the barrier-to-autointegration factor (BAF). The function of BAF is to facilitate the tethering of chromatin to the membrane of the nucleus, however in the case of necrosis, when BAF is phosphorylated, it will initiate the dissociation between the nuclear membrane and the condensed chromatin. [6] As a result, the nuclear membrane will collapse onto the condensed chromatin. Thus, the phosphorylation of BAF is a critical marker of necrotic pyknosis.

Significance of pyknosis

Original caption: Histopathological findings of the resected left adrenal gland (September 2009). a Gross appearance of the cut surface of the left adrenal tumor 3 cm in size showed the inferior surface to be necrotic. b-i Microscopic examination of the left adrenal tumor (b, d-f; hematoxylin and eosin staining. c, g-i; chromogranin A staining). Nontumoral adrenal gland in the right lower corner, and well-encapsulated tumor in the remainder of the photograph (b). The tumor had a large area of coagulative necrosis in the center. The necrotic material contained morphologically ghost cells (d, e) and was immunohistochemically markedly positive for chromogranin A (c, g, h). There were numerous hemosiderin-laden macrophages and histiocytes accompanied by vascular proliferation in the region adjacent to the area of necrosis (e, h). The viable region along the periphery of the tumor contained numerous cells undergoing pyknosis (f), and the cytoplasm of the tumor cells was positive for chromogranin A staining (i) Histopathology of a pheochromocytoma with coagulative necrosis, with immunostaining.jpg
Original caption: Histopathological findings of the resected left adrenal gland (September 2009). a Gross appearance of the cut surface of the left adrenal tumor 3 cm in size showed the inferior surface to be necrotic. b−i Microscopic examination of the left adrenal tumor (b, d−f; hematoxylin and eosin staining. c, g−i; chromogranin A staining). Nontumoral adrenal gland in the right lower corner, and well-encapsulated tumor in the remainder of the photograph (b). The tumor had a large area of coagulative necrosis in the center. The necrotic material contained morphologically ghost cells (d, e) and was immunohistochemically markedly positive for chromogranin A (c, g, h). There were numerous hemosiderin-laden macrophages and histiocytes accompanied by vascular proliferation in the region adjacent to the area of necrosis (e, h). The viable region along the periphery of the tumor contained numerous cells undergoing pyknosis (f), and the cytoplasm of the tumor cells was positive for chromogranin A staining (i)

Pyknosis is a stage in the apoptotic or necrotic cell death pathways. It is an important stage that involves fragmentation and condensation of damaged DNA/chromatin. Without it, the apoptotic or necrotic cell death pathways would be interrupted. This disruption, in turn, may prompt the improper destruction or removal of a cell with damaged elements as well as other related issues. These issues include cell accumulation and uncontrolled cell growth, which results in the formation of cancerous and abnormal tissue masses known as tumors. Therefore, being able to observe or identify when a cell is pyknotic (which may indicate that the cell is undergoing apoptosis or necrosis) and if it then successfully undergoes apoptosis or necrosis, may be crucial in determining if the cell will undergo uncontrolled cell growth and contribute to the formation of a tumor.

Techniques for detecting or observing pyknosis in cells

Various techniques are used to detect/observe pyknosis. These techniques also help to differentiate between apoptotic or necrotic cells. The techniques are identified and described as follows:

Cellular staining

When stains and dyes are applied to locate pyknotic cells in a tissue sample, the cell becomes easily identifiable. The stains/dyes target the nuclear and blebbed fragments of a pyknotic cell, making them dark (light contrast) and more readily seen when the sample is placed under a light microscope. Fluorescence microscopy and flow cytometry also use staining (fluorescent stains) to target the DNA/nuclear fragments of cells. The fluorescent staining creates a contrast between normal cell DNA and pyknotic cell DNA, because pyknotic cell nuclear material is condensed.

Apoptotic DNA laddering visualized in agarose gel. DNA from cells treated with an apoptotic inducing substance (left). A 1 kb marker (middle). Untreated cell DNA (right) Apoptotic DNA Laddering.png
Apoptotic DNA laddering visualized in agarose gel. DNA from cells treated with an apoptotic inducing substance (left). A 1 kb marker (middle). Untreated cell DNA (right)

Gel electrophoresis

Gel electrophoresis is a standard technique that is frequently used to visualize DNA fragmentation (forming a ladder-like image on the gel), which is a characteristic of apoptosis and is associated with nuclear condensation (which characterizes pyknosis). Therefore, when referring to apoptosis, this technique is known as DNA laddering. Gel electrophoresis is also used to visualize the random DNA fragmentation of necrosis, which forms a smear on the gel.

Assays to detect DNA fragmentation or condensation

Various assays of DNA fragmentation or condensation include the APO single-stranded DNA (ssDNA) assay which detects damaged DNA of cells undergoing apoptosis or necrosis, TUNEL assay which is used to locally find DNA strand breaks (DSBs), and ISEL. [8]

ISEL (in-situ labeling technique) is a labeling/tagging technique of apoptotic or necrotic cells. [8] ISEL specifically targets unfragmented DNA that has condensed into a nucleosome structure. [8]

The APO ssDNA assay detects apoptotic cells by using an antibody that specifically binds to the ssDNA, which is accumulated during apoptosis as a result of DNA fragmentation. [8] Therefore, the presence of ssDNA is an indicator of DNA damage in the apoptotic cell. For the assay process, cells are fixed (with e.g., formamide), and these cells then undergo incubation (at a predetermined temperature), which subjects the DNA to thermal denaturation and exposes the ssDNA. [8] Next, the cells are incubated with an ssDNA-specific antibody along with a fluorescently labeled secondary antibody. [8] The fluorescence amounts as a measure of apoptosis which can then be quantified using flow cytometry.

The TUNEL assay, otherwise known as the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, is a technique that measures DNA damage and breakage during apoptosis. [8] During apoptosis, DNA fragmentation exposes numerous 3’OH ends, that are labeled with modified deoxy-uridine triphosphate (dUTP) by the TUNEL reaction. [8] Then, this modified dUTP can be identified with specific fluorescent antibodies which can identify modified nucleotides or by using tagged nucleotides themselves. [8] Flow cytometry can then be used to quantify fluorescence intensity, and thus provide a measure of apoptosis.

Detection methods of caspase activity

As mentioned above, caspase proteins, which are protease enzymes, promote DNA condensation and fragmentation via the caspase (or proteolytic) cascade. These caspase proteins include, for example, caspase 9, caspase 6, caspase 7, and caspase 3. The caspase cascade directly activates caspase-activated DNase (CAD) which initiates DNA fragmentation into smaller pieces resulting in chromatin condensation. The biochemical techniques used to detect caspase activity include ELISA and fluorometric and colorimetric assays. [8]

See also

Related Research Articles

<span class="mw-page-title-main">Apoptosis</span> Form of programmed cell death

Apoptosis is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. The average adult human loses 50 to 70 billion cells each day due to apoptosis. For the average human child between 8 and 14 years old, each day the approximate loss is 20 to 30 billion cells.

<span class="mw-page-title-main">Necrosis</span> Unprogrammed cell death caused by external cell injury

Necrosis is a form of cell injury which results in the premature death of cells in living tissue by autolysis. The term "necrosis" came about in the mid-19th century and is commonly attributed to German pathologist Rudolf Virchow, who is often regarded as one of the founders of modern pathology. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated digestion of cell components. In contrast, apoptosis is a naturally occurring programmed and targeted cause of cellular death. While apoptosis often provides beneficial effects to the organism, necrosis is almost always detrimental and can be fatal.

<span class="mw-page-title-main">Lamin</span> Fibrous proteins

Lamins, also known as nuclear lamins are fibrous proteins in type V intermediate filaments, providing structural function and transcriptional regulation in the cell nucleus. Nuclear lamins interact with inner nuclear membrane proteins to form the nuclear lamina on the interior of the nuclear envelope. Lamins have elastic and mechanosensitive properties, and can alter gene regulation in a feedback response to mechanical cues. Lamins are present in all animals but are not found in microorganisms, plants or fungi. Lamin proteins are involved in the disassembling and reforming of the nuclear envelope during mitosis, the positioning of nuclear pores, and programmed cell death. Mutations in lamin genes can result in several genetic laminopathies, which may be life-threatening.

<span class="mw-page-title-main">Karyorrhexis</span> Destructive fragmentation of the nucleus of a dying cell

Karyorrhexis is the destructive fragmentation of the nucleus of a dying cell whereby its chromatin is distributed irregularly throughout the cytoplasm. It is usually preceded by pyknosis and can occur as a result of either programmed cell death (apoptosis), cellular senescence, or necrosis.

<span class="mw-page-title-main">Karyolysis</span>

Karyolysis, and λύσις lysis from λύειν lyein, "to separate") is the complete dissolution of the chromatin of a dying cell due to the enzymatic degradation by endonucleases. The whole cell will eventually stain uniformly with eosin after karyolysis. It is usually associated with karyorrhexis and occurs mainly as a result of necrosis, while in apoptosis after karyorrhexis the nucleus usually dissolves into apoptotic bodies.

<span class="mw-page-title-main">DNA laddering</span>

DNA laddering is a feature that can be observed when DNA fragments, resulting from Apoptosis DNA fragmentation are visualized after separation by gel electrophoresis the first described in 1980 by Andrew Wyllie at the University Edinburgh medical school DNA fragments can also be detected in cells that underwent necrosis, but when these DNA fragments after separation are subjected to gel electrophoresis no clear "ladder" pattern is apparent.

Cell damage is a variety of changes of stress that a cell suffers due to external as well as internal environmental changes. Amongst other causes, this can be due to physical, chemical, infectious, biological, nutritional or immunological factors. Cell damage can be reversible or irreversible. Depending on the extent of injury, the cellular response may be adaptive and where possible, homeostasis is restored. Cell death occurs when the severity of the injury exceeds the cell's ability to repair itself. Cell death is relative to both the length of exposure to a harmful stimulus and the severity of the damage caused. Cell death may occur by necrosis or apoptosis.

<span class="mw-page-title-main">Apoptosis-inducing factor</span> Protein family

Apoptosis inducing factor is involved in initiating a caspase-independent pathway of apoptosis by causing DNA fragmentation and chromatin condensation. Apoptosis inducing factor is a flavoprotein. It also acts as an NADH oxidase. Another AIF function is to regulate the permeability of the mitochondrial membrane upon apoptosis. Normally it is found behind the outer membrane of the mitochondrion and is therefore secluded from the nucleus. However, when the mitochondrion is damaged, it moves to the cytosol and to the nucleus. Inactivation of AIF leads to resistance of embryonic stem cells to death following the withdrawal of growth factors indicating that it is involved in apoptosis.

Fragmentation describes the process of splitting into several pieces or fragments. In cell biology, fragmentation is useful for a cell during both DNA cloning and apoptosis. DNA cloning is important in asexual reproduction or creation of identical DNA molecules, and can be performed spontaneously by the cell or intentionally by laboratory researchers. Apoptosis is the programmed destruction of cells, and the DNA molecules within them, and is a highly regulated process. These two ways in which fragmentation is used in cellular processes describe normal cellular functions and common laboratory procedures performed with cells. However, problems within a cell can sometimes cause fragmentation that results in irregularities such as red blood cell fragmentation and sperm cell DNA fragmentation.

<span class="mw-page-title-main">ENDOG</span> Protein-coding gene in the species Homo sapiens

Endonuclease G, mitochondrial is an enzyme that in humans is encoded by the ENDOG gene. This protein primarily participates in caspase-independent apoptosis via DNA degradation when translocating from the mitochondrion to nucleus under oxidative stress. As a result, EndoG has been implicated in cancer, aging, and neurodegenerative diseases such as Parkinson's disease (PD). Regulation of its expression levels thus holds potential to treat or ameliorate those conditions.

<span class="mw-page-title-main">Apoptotic DNA fragmentation</span> Cleavage of DNA into tiny pieces during apoptosis

Apoptotic DNA fragmentation is a key feature of apoptosis, a type of programmed cell death. Apoptosis is characterized by the activation of endogenous endonucleases, particularly the caspase-3 activated DNase (CAD), with subsequent cleavage of nuclear DNA into internucleosomal fragments of roughly 180 base pairs (bp) and multiples thereof (360, 540 etc.). The apoptotic DNA fragmentation is being used as a marker of apoptosis and for identification of apoptotic cells either via the DNA laddering assay, the TUNEL assay, or the by detection of cells with fractional DNA content ("sub G1 cells") on DNA content frequency histograms e.g. as in the Nicoletti assay.

<span class="mw-page-title-main">ADP/ATP translocase 4</span> Protein-coding gene in the species Homo sapiens

ADP/ATP translocase 4 (ANT4) is an enzyme that in humans is encoded by the SLC25A31 gene on chromosome 4. This enzyme inhibits apoptosis by catalyzing ADP/ATP exchange across the mitochondrial membranes and regulating membrane potential. In particular, ANT4 is essential to spermatogenesis, as it imports ATP into sperm mitochondria to support their development and survival. Outside this role, the SLC25AC31 gene has not been implicated in any human disease.

<span class="mw-page-title-main">Caspase-activated DNase</span> Protein found in humans

Caspase-activated DNase (CAD) or DNA fragmentation factor subunit beta is a protein that in humans is encoded by the DFFB gene. It breaks up the DNA during apoptosis and promotes cell differentiation. It is usually an inactive monomer inhibited by ICAD. This is cleaved before dimerization.

Ischemic cell death, or oncosis, is a form of accidental cell death. The process is characterized by an ATP depletion within the cell leading to impairment of ionic pumps, cell swelling, clearing of the cytosol, dilation of the endoplasmic reticulum and golgi apparatus, mitochondrial condensation, chromatin clumping, and cytoplasmic bleb formation. Oncosis refers to a series of cellular reactions following injury that precedes cell death. The process of oncosis is divided into three stages. First, the cell becomes committed to oncosis as a result of damage incurred to the plasma membrane through toxicity or ischemia, resulting in the leak of ions and water due to ATP depletion. The ionic imbalance that occurs subsequently causes the cell to swell without a concurrent change in membrane permeability to reverse the swelling. In stage two, the reversibility threshold for the cell is passed and the cell becomes committed to cell death. During this stage the membrane becomes abnormally permeable to trypan blue and propidium iodide, indicating membrane compromise. The final stage is cell death and removal of the cell via phagocytosis mediated by an inflammatory response.

<span class="mw-page-title-main">Necroptosis</span> Programmed form of necrosis, or inflammatory cell death

Necroptosis is a programmed form of necrosis, or inflammatory cell death. Conventionally, necrosis is associated with unprogrammed cell death resulting from cellular damage or infiltration by pathogens, in contrast to orderly, programmed cell death via apoptosis. The discovery of necroptosis showed that cells can execute necrosis in a programmed fashion and that apoptosis is not always the preferred form of cell death. Furthermore, the immunogenic nature of necroptosis favors its participation in certain circumstances, such as aiding in defence against pathogens by the immune system. Necroptosis is well defined as a viral defense mechanism, allowing the cell to undergo "cellular suicide" in a caspase-independent fashion in the presence of viral caspase inhibitors to restrict virus replication. In addition to being a response to disease, necroptosis has also been characterized as a component of inflammatory diseases such as Crohn's disease, pancreatitis, and myocardial infarction.

Immunogenic cell death is any type of cell death eliciting an immune response. Both accidental cell death and regulated cell death can result in immune response. Immunogenic cell death contrasts to forms of cell death that do not elicit any response or even mediate immune tolerance.

<span class="mw-page-title-main">Paraptosis</span> Type of programmed cell death distinct from apoptosis and necrosis

Paraptosis is a type of programmed cell death, morphologically distinct from apoptosis and necrosis. The defining features of paraptosis are cytoplasmic vacuolation, independent of caspase activation and inhibition, and lack of apoptotic morphology. Paraptosis lacks several of the hallmark characteristics of apoptosis, such as membrane blebbing, chromatin condensation, and nuclear fragmentation. Like apoptosis and other types of programmed cell death, the cell is involved in causing its own death, and gene expression is required. This is in contrast to necrosis, which is non-programmed cell death that results from injury to the cell.

<span class="mw-page-title-main">BinCARD</span> Protein-coding gene in the species Homo sapiens

Bcl10-interacting CARD protein, also known as BinCARD, is a protein that in humans is encoded by the C9orf89 gene on chromosome 9. BinCARD is a member of the death-domain superfamily and contains a caspase recruitment domain (CARD). This protein regulates apoptosis and the immune response by inhibiting Bcl10, thus implicating it in diseases stemming from Bcl10 dysfunction.

<span class="mw-page-title-main">FASTKD2</span> Protein-coding gene in the species Homo sapiens

FAST kinase domain-containing protein 2 (FASTKD2) is a protein that in humans is encoded by the FASTKD2 gene on chromosome 2. This protein is part of the FASTKD family, which is known for regulating the energy balance of mitochondria under stress. FASTKD2 has been implicated in mitochondrial encephalomyopathy, breast cancer, and prostate cancer.

<span class="mw-page-title-main">FAM162A</span> Protein-coding gene in the species Homo sapiens

Human growth and transformation-dependent protein (HGTD-P), also called E2-induced gene 5 protein (E2IG5), is a protein that in humans is encoded by the FAM162A gene on chromosome 3. This protein promotes intrinsic apoptosis in response to hypoxia via interactions with hypoxia-inducible factor-1α (HIF-1α). As a result, it has been associated with cerebral ischemia, myocardial infarction, and various cancers.

References

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