Pyknosis
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Pyknosis, or karyopyknosis, is the irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis<ref>Template:Cite book</ref> or apoptosis.<ref name="Kroemer_2009">Template:Cite journal</ref> It is typically followed by karyorrhexis, the fragmentation of the nucleus.
The term derives from Ancient Greek Template:Lang meaning "thick, closed or condensed". Pyknosis also occurs as part of normal cell maturation in certain blood cells.
Occurrence
In erythropoiesis, the maturing metarubricyte condenses its nucleus before expelling it to form a reticulocyte. In granulopoiesis, the developing neutrophil condenses its nucleus into several connected lobes, which remain in the cell until its death.
Pyknotic nuclei are also commonly seen in the zona reticularis of the adrenal gland and in the keratinocytes of the outermost layer of parakeratinised epithelium.
Mechanism
Pyknosis represents irreversible nuclear condensation in a cell undergoing apoptosis or necrosis.<ref>Template:Cite journal</ref> Two main types are described: nucleolytic pyknosis, which occurs during apoptosis (programmed cell death), and anucleolytic pyknosis, which occurs during necrosis.<ref name="Liu_2023">Template:Cite book</ref>
Necrosis is a regulated form of cell death triggered by toxins, infections, or other acute stressors.<ref name="Liu_2023" /> Such stress leads to swelling and morphological changes of cellular organelles, ultimately compromising the cell membrane.<ref name="Liu_2023" />
Pyknosis is characterized by nuclear shrinkage and dense chromatin, which ultimately fragments in karyorrhexis.<ref name="Valenciano_2014">Template:Cite book</ref> Karyorrhexis refers to the subsequent fragmentation of this condensed nucleus and cleavage of chromatin.<ref name="Valenciano_2014" />
In apoptosis and necrosis
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.<ref name="Hou_2016">Template:Cite journal</ref> 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.<ref name="Hou_2016" /> 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.<ref name="Hou_2016" /> Consequently, pyknosis can be distinguished into two types, nucleolytic pyknosis (apoptotic cells) and anucleolytic pyknosis (necrotic cells).
Types
Nucleolytic
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.<ref name="Liu_2023" /> 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).<ref name="Liu_2023" /> 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.<ref name="Liu_2023" />
Anucleolytic
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).<ref name="Liu_2023" /> 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.<ref name="Hou_2016" /> As a result, the nuclear membrane will collapse onto the condensed chromatin. Thus, the phosphorylation of BAF is a critical marker of necrotic pyknosis.
Detection
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.
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.
DNA assays
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.<ref name="Kari_2022">Template:Cite journal</ref>
ISEL (in-situ labeling technique) is a labeling/tagging technique of apoptotic or necrotic cells.<ref name="Kari_2022" /> ISEL specifically targets unfragmented DNA that has condensed into a nucleosome structure.<ref name="Kari_2022" />
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.<ref name="Kari_2022" /> 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.<ref name="Kari_2022" /> Next, the cells are incubated with an ssDNA-specific antibody along with a fluorescently labeled secondary antibody.<ref name="Kari_2022" /> 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.<ref name="Kari_2022" /> During apoptosis, DNA fragmentation exposes numerous 3’OH ends, that are labeled with modified deoxy-uridine triphosphate (dUTP) by the TUNEL reaction.<ref name="Kari_2022" /> Then, this modified dUTP can be identified with specific fluorescent antibodies which can identify modified nucleotides or by using tagged nucleotides themselves.<ref name="Kari_2022" /> Flow cytometry can then be used to quantify fluorescence intensity, and thus provide a measure of apoptosis.
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.<ref name="Kari_2022" />
Clinical significance
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.