Nocodazole
Encyclopedia
Nocodazole is an anti-neoplastic agent
which exerts its effect in cells by interfering with the polymer
ization of microtubule
s. Microtubules are one type of fibre which constitutes the cytoskeleton
, and the dynamic microtubule network has several important roles in the cell, including vesicular transport, forming the mitotic spindle
and in cytokinesis
.
Several drugs including vincristine
and colcemid
are similar to nocodazole in that they interfere with microtubule polymerisation.
experiments as a control: for example, some dominant negative Rho
small GTPase
s cause a similar effect as nocodazole, and constitutively activated mutants often reverse or negate the effect.
Nocodazole is frequently used in cell biology laboratories to synchronize the cell division cycle
. Cells treated with nocodazole arrest with a G2
- or M
-phase DNA content when analysed by flow cytometry
. Microscopy of nocodazole-treated cells shows that they do enter mitosis
but cannot form metaphase
spindles
because microtubules (of which the spindles are made) cannot polymerise. The absence of microtubule attachment to kinetochore
s activates the spindle assembly checkpoint
, causing the cell to arrest in prometaphase
. For cell synchronization experiments, nocodazole is usually used at a concentration of 40-100 ng/mL of culture medium for a duration of 12-18 hrs. Prolonged arrest of cells in mitosis due to nocodazole treatment typically results in cell death by apoptosis
.
Another standard cell biological application of nocodazole is to induce the formation of Golgi
ministacks in eukaryotic cells. The perinuclear structural organization of the Golgi apparatus
in eukaryotes is dependent on microtubule trafficking, but disrupting the trafficking of Golgi elements from the endoplasmic reticulum treatment with nocodazole (33 uM for 3 hours works) induces numerous Golgi elements to form adjacent to ER exit sites. These functional Golgi ministacks remain distributed about the cell, unable to track forward to form a perinuclear Golgi since nocodazole has depolymerized the microtubules.
Chemical compound
A chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
which exerts its effect in cells by interfering with the polymer
Polymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
ization of microtubule
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...
s. Microtubules are one type of fibre which constitutes the cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
, and the dynamic microtubule network has several important roles in the cell, including vesicular transport, forming 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...
and in cytokinesis
Cytokinesis
Cytokinesis is the process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells. It usually initiates during the late stages of mitosis, and sometimes meiosis, splitting a binucleate cell in two, to ensure that chromosome number is maintained from one generation...
.
Several drugs including vincristine
Vincristine
Vincristine , formally known as leurocristine, sometimes abbreviated "VCR", is a vinca alkaloid from the Catharanthus roseus , formerly Vinca rosea and hence its name. It is a mitotic inhibitor, and is used in cancer chemotherapy.-Mechanism:Tubulin is a structural protein that polymerizes to...
and colcemid
Colcemid
Used in cytogenetics, colcemid, also known as demecolcine, is related to colchicine but it is less toxic. It depolymerises microtubules and limits microtubule formation , thus arresting cells in metaphase and allowing cell harvest and karyotyping to be performed.-Mechanism of Action:Colcemid is a...
are similar to nocodazole in that they interfere with microtubule polymerisation.
Use in cell biology research
As nocodazole affects the cytoskeleton, it is often used in cell biologyCell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
experiments as a control: for example, some dominant negative Rho
Rho
Rho is the 17th letter of the Greek alphabet. In the system of Greek numerals, it has a value of 100. It is derived from Semitic resh "head"...
small GTPase
Small GTPase
Small GTPases are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate . They are a form of G-proteins found in the cytosol which are homologous to the alpha subunit of heterotrimeric G-proteins, but unlike the alpha subunit of G proteins, a small GTPase can function...
s cause a similar effect as nocodazole, and constitutively activated mutants often reverse or negate the effect.
Nocodazole is frequently used in cell biology laboratories to synchronize the cell division cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
. Cells treated with nocodazole arrest with a G2
G2 phase
G2 phase is the 3rd and final subphase of Interphase in the cell cycle directly preceding Mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated...
- or M
Mitosis
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...
-phase DNA content when analysed by flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
. Microscopy of nocodazole-treated cells shows that they do enter mitosis
Mitosis
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...
but cannot form metaphase
Metaphase
Metaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
spindles
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...
because microtubules (of which the spindles are made) cannot polymerise. The absence of microtubule attachment to kinetochore
Kinetochore
The kinetochore is the protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart....
s activates the spindle assembly checkpoint
Spindle checkpoint
In order to preserve one cell's identity and its proper functioning, it is necessary to maintain constant the appropriate number of chromosomes after each cell division...
, causing the cell to arrest in prometaphase
Prometaphase
Prometaphase is the phase of mitosis following prophase and preceding metaphase, in eukaryotic somatic cells. In Prometaphase, The nuclear envelope breaks into fragments and disappears. The tiny nucleolus inside the nuclear envolope, also dissolves. Microtubules emerging from the centrosomes at the...
. For cell synchronization experiments, nocodazole is usually used at a concentration of 40-100 ng/mL of culture medium for a duration of 12-18 hrs. Prolonged arrest of cells in mitosis due to nocodazole treatment typically results in cell death by apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
.
Another standard cell biological application of nocodazole is to induce the formation of Golgi
Golgi
Golgi may refer to:*Camillo Golgi , Italian physician and scientist after which the following terms are named:**Golgi apparatus , an organelle in the eukaryotic cell...
ministacks in eukaryotic cells. The perinuclear structural organization of the Golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....
in eukaryotes is dependent on microtubule trafficking, but disrupting the trafficking of Golgi elements from the endoplasmic reticulum treatment with nocodazole (33 uM for 3 hours works) induces numerous Golgi elements to form adjacent to ER exit sites. These functional Golgi ministacks remain distributed about the cell, unable to track forward to form a perinuclear Golgi since nocodazole has depolymerized the microtubules.