Katanin
Encyclopedia
Katanin is a microtubule
-severing AAA protein. It is named after the Japanese sword, katana
. Katanin is a heterodimeric protein first discovered in sea urchin
s. It contains a 60 kDa ATPase
subunit
, which functions to sever microtubules. This subunit requires ATP
and the presence of microtubules for activation. The second 80 kDA subunit regulates the activity of the ATPase and localizes the protein to the centrosomes. Electron microscopy shows that katanin forms 14–16 nm rings in its active oligomer
ized state on the walls of microtubules:
has revealed that microtubule protofilaments change from a straight to a curved conformation upon GTP
hydrolysis
of β-tubulin
. However, when these protofilaments are part of a polymerized microtubule, the stabilizing interactions created by the surrounding lattice lock subunits into a straight conformation, even after GTP hydrolysis
. In order to disrupt these stable interactions, katanin, once activated by ATP, oligomerizes into a ring structure on the microtubule wall - oligomerization increases the affinity of katanin for microtubules and stimulates its ATPase activity. Once this structure is formed, each katanin protein hydrolyzes an ATP molecule, and subsequently undergoes a conformational change that puts mechanical strain on the tubulin subunits, which destabilizes their interactions within the microtubule lattice (13 longitudinal bonds must be broken). The conformational change also decreases the affinity of katanin for tubulin as well as for other katanin proteins, which leads to disassembly of the katanin ring structure, and recycling of the individual inactivated proteins.
The severing of microtubules by katanin is regulated by nucleotide exchange factor
s, which can exchange ADP
with ATP, protective microtubule-associated proteins (MAPs), and the p80 subunit (p60 severs microtubules much better in the presence of p80). These mechanisms have different consequences, depending on where in the cell
they are activated or disrupted. For example, allowing katanin-mediated severing at the centrosome releases microtubules for free movement. In one experiment, anti-katanin antibodies
were injected into a cell, causing a large accumulation of microtubules around the centrosome and inhibition of microtubule outgrowth. Therefore, katanin-mediated severing may serve to maintain organization in the cytoplasm
by promoting microtubule disassembly and efficient movement. During cell division, severing at the spindle pole produces free microtubule ends and allows poleward flux of tubulin and retraction of the microtubule. Severing microtubules in the cytoplasm facilitates treadmilling
and mobility, which is important during development.
and meiosis
. It has been shown that katanin is responsible for severing microtubules during M-phase in Xenopus laevis. The disassembly of microtubules from their interphase
structures is necessary to prepare the cell and the mitotic spindle
for cell division. This regulation is indirect: MAP proteins, which protect the microtubules from being severed during interphase, dissociate and allow katanin to act. In addition, katanin is responsible for severing microtubules at the mitotic spindles when disassembly is required to segregate sister chromatids during anaphase
.
Similar results have been obtained in relation to katanin’s activity during meiosis in C. elegans
. It was reported that Mei-1 and Mei-2 to encode similar proteins to the p60 and p80 subunits of katanin. Using antibodies, these two proteins were found to localize at the ends of microtubules in the meiotic spindle, and, when expressed in HeLa cells, these proteins initiated microtubule severing. These findings indicate that katanin serves a similar purpose in both mitosis and meiosis in segregating chromatids toward the spindle poles.
al growth, and, thus, katanin must be carefully regulated for proper neural development. In particular, severing microtubules in specific cellular spaces allows fragments to test various routes of growth. Katanin has proved necessary in this task. An experiment using time-lapse digital imaging of fluorescently labeled tubulin demonstrated that axon growth cones pause, and microtubules fragment, at sites of branching during neural development.
A similar experiment using fluorescently labeled tubulin observed local microtubule fragmentation in newt
lung
cell lamellipodia
during developmental migration, in which the fragments run perpendicular to the advancing cell membrane to aid exploration. The local nature of both fragmentation events likely indicates regulation by katanin because it can be concentrated in specific cellular regions. This is supported by a study that demonstrated that the Fra2 mutation, which affects a katanin orthologue in Arabidopsis thaliana
, leads to an aberrant disposition of cellulose
microfibrils along the developing cell wall in these plants. This mutation produced a phenotype
with reduced cell elongation, which suggests katanin’s significance in development across a wide range of organisms.
and even modest levels of it can cause significant microtubule depletion. But microtubules need to be severed throughout other compartments of the neuron so that sufficient numbers of microtubules can undergo rapid transport.
In the nervous system, the ratio of the two subunits is dramatically different than other organs of the body. So it is important to be able to regulate the ratio to control microtubule severing. The monomer p80 is found in all the compartments of the neuron, which means its function cannot be solely to target katanin. The p80 katanin has multiple domains with different functions. One domain targets the centrosome, another augments microtubule severing by the p60 katanin, and the last suppresses microtubule severing. The abundance of katanin in the neurons show they can move along the axon. There is breakage of microtubules at the axonal branch points and in the growth cones of the neurons. The distribution of katanin in the neuron helps understand the phenomenon for regulating microtubule length and number, as well as releasing the microtubules from the centrosome.
Katanin is believed to be regulated by the phosphorylation
of other proteins. Microtubules break into fibroblasts after slight bending. But, when katanin is present, the bending can lead to breakage because it enhances the access of katanin to the lattice.
, which contains highly organized cellulose, the orientation of which is affected by microtubules that serve to guide the deposition of forming fibers. The orientation of the cellulose microfibrils within the cell wall is determined by the microtubules, which are aligned perpendicular to the major axis of cell expansion. Because plant cells lack traditional centrosomes, katanin accumulates at the nuclear envelope
during pre-prophase and prophase
, where the spindle microtubules are forming.
During cell elongation, microtubules must adjust their orientation constantly to keep up with the increasing cell length. This constant change in microtubule organization was proposed to be performed by the rapid disassembly, assembly, and translocation of microtubules. Recently, mutations in the plant katanin homologue
have been shown to alter transitions in microtubule organization, which, in turn, cause impairments in the proper deposition of cellulose and hemicellulose
. This is presumed to be caused by the plant cell's lack of ability to regulate microtubule lengths.
There is no homologue for the p80 katanin regulatory subunit. Therefore, a His-tagged At-p60 was made to describe its functions in plants. The His-At-p60 can sever microtubules in vitro in the presence of ATP. It directly interacts with microtubules in co-sedimentation assays. The ATPase activity was stimulated in a non-hyperbolic way. ATP hydrolysis is stimulated at a low tubulin/At-p60 ratio and inhibited at higher ratios. The low ratios favor the katanin subunit interactions, whereas the high ratios show impairment. The At-p60 can oligomerize like the ones in animals. The At-p60 interacts directly with microtubules, whereas the animal p60 bind via their N-termini
. The N-terminal part of p60 is not well conserved between the plant and animal kingdoms.
Microtubule
Microtubules are a component of the cytoskeleton. These rope-like polymers of tubulin can grow as long as 25 micrometers and are highly dynamic. The outer diameter of microtubule is about 25 nm. Microtubules are important for maintaining cell structure, providing platforms for intracellular...
-severing AAA protein. It is named after the Japanese sword, katana
Katana
A Japanese sword, or , is one of the traditional bladed weapons of Japan. There are several types of Japanese swords, according to size, field of application and method of manufacture.-Description:...
. Katanin is a heterodimeric protein first discovered in sea urchin
Sea urchin
Sea urchins or urchins are small, spiny, globular animals which, with their close kin, such as sand dollars, constitute the class Echinoidea of the echinoderm phylum. They inhabit all oceans. Their shell, or "test", is round and spiny, typically from across. Common colors include black and dull...
s. It contains a 60 kDa ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
subunit
Protein subunit
In structural biology, a protein subunit or subunit protein is a single protein molecule that assembles with other protein molecules to form a protein complex: a multimeric or oligomeric protein. Many naturally occurring proteins and enzymes are multimeric...
, which functions to sever microtubules. This subunit requires ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
and the presence of microtubules for activation. The second 80 kDA subunit regulates the activity of the ATPase and localizes the protein to the centrosomes. Electron microscopy shows that katanin forms 14–16 nm rings in its active oligomer
Oligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
ized state on the walls of microtubules:
Mechanism and regulation of microtubule length
Structural analysis using X-ray crystallographyX-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
has revealed that microtubule protofilaments change from a straight to a curved conformation upon GTP
Guanosine triphosphate
Guanosine-5'-triphosphate is a purine nucleoside triphosphate. It can act as a substrate for the synthesis of RNA during the transcription process...
hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of β-tubulin
Tubulin
Tubulin is one of several members of a small family of globular proteins. The most common members of the tubulin family are α-tubulin and β-tubulin, the proteins that make up microtubules. Each has a molecular weight of approximately 55 kiloDaltons. Microtubules are assembled from dimers of α- and...
. However, when these protofilaments are part of a polymerized microtubule, the stabilizing interactions created by the surrounding lattice lock subunits into a straight conformation, even after GTP hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
. In order to disrupt these stable interactions, katanin, once activated by ATP, oligomerizes into a ring structure on the microtubule wall - oligomerization increases the affinity of katanin for microtubules and stimulates its ATPase activity. Once this structure is formed, each katanin protein hydrolyzes an ATP molecule, and subsequently undergoes a conformational change that puts mechanical strain on the tubulin subunits, which destabilizes their interactions within the microtubule lattice (13 longitudinal bonds must be broken). The conformational change also decreases the affinity of katanin for tubulin as well as for other katanin proteins, which leads to disassembly of the katanin ring structure, and recycling of the individual inactivated proteins.
The severing of microtubules by katanin is regulated by nucleotide exchange factor
Nucleotide exchange factor
Nucleotide exchange factors are proteins that stimulate the exchange of nucleoside diphosphates for nucleoside triphosphates bound to other proteins.-Function:...
s, which can exchange ADP
Adenosine diphosphate
Adenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....
with ATP, protective microtubule-associated proteins (MAPs), and the p80 subunit (p60 severs microtubules much better in the presence of p80). These mechanisms have different consequences, depending on where in the cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
they are activated or disrupted. For example, allowing katanin-mediated severing at the centrosome releases microtubules for free movement. In one experiment, anti-katanin antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
were injected into a cell, causing a large accumulation of microtubules around the centrosome and inhibition of microtubule outgrowth. Therefore, katanin-mediated severing may serve to maintain organization in the cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
by promoting microtubule disassembly and efficient movement. During cell division, severing at the spindle pole produces free microtubule ends and allows poleward flux of tubulin and retraction of the microtubule. Severing microtubules in the cytoplasm facilitates treadmilling
Treadmilling
Treadmilling is a phenomenon observed in many cellular cytoskeletal filaments, especially in actin filaments and microtubules. It occurs when one end of a filament grows in length while the other end shrinks resulting in a section of filament seemingly "moving" across a stratum or the cytosol...
and mobility, which is important during development.
Role in cell division
Katanin-mediated microtubule severing is an important step in mitosisMitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
and meiosis
Meiosis
Meiosis is a special type of cell division necessary for sexual reproduction. The cells produced by meiosis are gametes or spores. The animals' gametes are called sperm and egg cells....
. It has been shown that katanin is responsible for severing microtubules during M-phase in Xenopus laevis. The disassembly of microtubules from their interphase
Interphase
Interphase is the phase of the cell cycle in which the cell spends the majority of its time and performs the majority of its purposes including preparation for cell division. In preparation for cell division, it increases its size and makes a copy of its DNA...
structures is necessary to prepare the cell and the mitotic spindle
Mitotic spindle
In cell biology, the spindle fibers are the structure that separates the chromosomes into the daughter cells during cell division. It is part of the cytoskeleton in eukaryotic cells...
for cell division. This regulation is indirect: MAP proteins, which protect the microtubules from being severed during interphase, dissociate and allow katanin to act. In addition, katanin is responsible for severing microtubules at the mitotic spindles when disassembly is required to segregate sister chromatids during anaphase
Anaphase
Anaphase, from the ancient Greek ἀνά and φάσις , is the stage of mitosis or meiosis when chromosomes move to opposite poles of the cell....
.
Similar results have been obtained in relation to katanin’s activity during meiosis in C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
. It was reported that Mei-1 and Mei-2 to encode similar proteins to the p60 and p80 subunits of katanin. Using antibodies, these two proteins were found to localize at the ends of microtubules in the meiotic spindle, and, when expressed in HeLa cells, these proteins initiated microtubule severing. These findings indicate that katanin serves a similar purpose in both mitosis and meiosis in segregating chromatids toward the spindle poles.
Role in development
Katanin is important in the development of many organisms. Both elimination and overexpression of katanin is deleterious to axonAxon
An axon is a long, slender projection of a nerve cell, or neuron, that conducts electrical impulses away from the neuron's cell body or soma....
al growth, and, thus, katanin must be carefully regulated for proper neural development. In particular, severing microtubules in specific cellular spaces allows fragments to test various routes of growth. Katanin has proved necessary in this task. An experiment using time-lapse digital imaging of fluorescently labeled tubulin demonstrated that axon growth cones pause, and microtubules fragment, at sites of branching during neural development.
A similar experiment using fluorescently labeled tubulin observed local microtubule fragmentation in newt
Newt
A newt is an aquatic amphibian of the family Salamandridae, although not all aquatic salamanders are considered newts. Newts are classified in the subfamily Pleurodelinae of the family Salamandridae, and are found in North America, Europe and Asia...
lung
Lung
The lung is the essential respiration organ in many air-breathing animals, including most tetrapods, a few fish and a few snails. In mammals and the more complex life forms, the two lungs are located near the backbone on either side of the heart...
cell lamellipodia
Lamellipodia
The lamellipodium is a cytoskeletal protein actin projection on the mobile edge of the cell. It contains a quasi-two-dimensional actin mesh; the whole structure propels the cell across a substrate...
during developmental migration, in which the fragments run perpendicular to the advancing cell membrane to aid exploration. The local nature of both fragmentation events likely indicates regulation by katanin because it can be concentrated in specific cellular regions. This is supported by a study that demonstrated that the Fra2 mutation, which affects a katanin orthologue in Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
, leads to an aberrant disposition of cellulose
Cellulose
Cellulose is an organic compound with the formula , a polysaccharide consisting of a linear chain of several hundred to over ten thousand β linked D-glucose units....
microfibrils along the developing cell wall in these plants. This mutation produced a phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
with reduced cell elongation, which suggests katanin’s significance in development across a wide range of organisms.
Function in neurons
Katanin is known to be abundant in the nervous systemNervous system
The nervous system is an organ system containing a network of specialized cells called neurons that coordinate the actions of an animal and transmit signals between different parts of its body. In most animals the nervous system consists of two parts, central and peripheral. The central nervous...
and even modest levels of it can cause significant microtubule depletion. But microtubules need to be severed throughout other compartments of the neuron so that sufficient numbers of microtubules can undergo rapid transport.
In the nervous system, the ratio of the two subunits is dramatically different than other organs of the body. So it is important to be able to regulate the ratio to control microtubule severing. The monomer p80 is found in all the compartments of the neuron, which means its function cannot be solely to target katanin. The p80 katanin has multiple domains with different functions. One domain targets the centrosome, another augments microtubule severing by the p60 katanin, and the last suppresses microtubule severing. The abundance of katanin in the neurons show they can move along the axon. There is breakage of microtubules at the axonal branch points and in the growth cones of the neurons. The distribution of katanin in the neuron helps understand the phenomenon for regulating microtubule length and number, as well as releasing the microtubules from the centrosome.
Katanin is believed to be regulated by the phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
of other proteins. Microtubules break into fibroblasts after slight bending. But, when katanin is present, the bending can lead to breakage because it enhances the access of katanin to the lattice.
Function in plants
Katanin is also found to have similar functions in higher plants. The form and structure of a plant cell is determined by the rigid cell wallCell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
, which contains highly organized cellulose, the orientation of which is affected by microtubules that serve to guide the deposition of forming fibers. The orientation of the cellulose microfibrils within the cell wall is determined by the microtubules, which are aligned perpendicular to the major axis of cell expansion. Because plant cells lack traditional centrosomes, katanin accumulates at the nuclear envelope
Nuclear envelope
A nuclear envelope is a double lipid bilayer that encloses the genetic material in eukaryotic cells. The nuclear envelope also serves as the physical barrier, separating the contents of the nucleus from the cytosol...
during pre-prophase and prophase
Prophase
Prophase, from the ancient Greek πρό and φάσις , is a stage of mitosis in which the chromatin condenses into a highly ordered structure called a chromosome in which the chromatin becomes visible. This process, called chromatin condensation, is mediated by the condensin complex...
, where the spindle microtubules are forming.
During cell elongation, microtubules must adjust their orientation constantly to keep up with the increasing cell length. This constant change in microtubule organization was proposed to be performed by the rapid disassembly, assembly, and translocation of microtubules. Recently, mutations in the plant katanin homologue
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
have been shown to alter transitions in microtubule organization, which, in turn, cause impairments in the proper deposition of cellulose and hemicellulose
Hemicellulose
A hemicellulose is any of several heteropolymers , such as arabinoxylans, present along with cellulose in almost all plant cell walls. While cellulose is crystalline, strong, and resistant to hydrolysis, hemicellulose has a random, amorphous structure with little strength...
. This is presumed to be caused by the plant cell's lack of ability to regulate microtubule lengths.
There is no homologue for the p80 katanin regulatory subunit. Therefore, a His-tagged At-p60 was made to describe its functions in plants. The His-At-p60 can sever microtubules in vitro in the presence of ATP. It directly interacts with microtubules in co-sedimentation assays. The ATPase activity was stimulated in a non-hyperbolic way. ATP hydrolysis is stimulated at a low tubulin/At-p60 ratio and inhibited at higher ratios. The low ratios favor the katanin subunit interactions, whereas the high ratios show impairment. The At-p60 can oligomerize like the ones in animals. The At-p60 interacts directly with microtubules, whereas the animal p60 bind via their N-termini
N-terminal end
The N-terminus refers to the start of a protein or polypeptide terminated by an amino acid with a free amine group . The convention for writing peptide sequences is to put the N-terminus on the left and write the sequence from N- to C-terminus...
. The N-terminal part of p60 is not well conserved between the plant and animal kingdoms.