Preparing for Cell Division: Part 1 Lecture Notes
Key words and terms
Cell division cycle; chromosome; chromatin; DNA; replication; condensation; mitosis; microtubule; centrosome; kinetochore; metaphase; anaphase; nucleus; complexity; assembly
The process of cell growth and division is introduced as a fundamental aspect of cell behavior. The difficulty of the process is expressed in terms of the complexity of any one cell, which makes it hard to synthesize and assemble all the parts necessary to make a second cell. Many macromolecules must be made and set in their correct places before a cell is ready to divide. All this synthetic activity occurs during “interphase”, the time between successive cell division.
Events of the cell growth and division cycle
One key event of interphase is the replication of the cell’s DNA, which occurs during the “S-phase”.
Another is the duplication of the centrosome, which usually occurs at about the beginning of S-phase. There is, however, a gap in time after the previous cell division is over and before S-phase begins (G1).
During G1, cells synthesize many macromolecules, like proteins that are essential for cell growth. It is also during this phase that most cells decide either to enter S-phase and divide again or to leave the cell division cycle and synthesize more specialized proteins, so they can “differentiate” into a form that will allow them to do a particular job, for example, become a neuron. This time of decision is important for cell behavior, because cells in a multicellular organism should divide only often enough to make new cells as needed.
Once S-phase has started, the cell is committed to divide again, but after S-phase is finished, there is usually yet another gap (G-2), allowing time for more RNA and protein synthesis before the cell is ready to go through the mechanical process of separating the now-double cell into two distinct objects, the “daughter cells”.
The logic of dividing the parts of a biochemically doubled cell
Most of the cell’s parts (organelles like mitochondria and macromolecular machines like ribosomes, etc.) are so numerous that they get no special attention at the time of cell division; roughly half will wind up in one cell and roughly half in the other, simply by chance.
Some cell parts are present in only one or a few copies, for example, each chromosome and the centrosome, a structure that helps to organize the microtubules of the cell’s cytoskeleton. Cells take special pains to make sure that each daughter cells gets one of each of these objects, so it will have all that it needs to grow and divide again. To achieve reliable separation of such structures, the cell builds a special machine that is designed to deliver one copy of each structure to each of the two daughter cells. This machine is called the “mitotic spindle”.
The mitotic spindle is built largely from microtubules and the many proteins that associate with them. In most cells the spindle microtubules are initiated by the already duplicated centrosomes, so the structure that forms has two, essentially identical parts. These microtubules grow into the space where the chromosomes reside, and some of them encounter a specialization found on each chromosome called a “kinetochore”, to which they bind, allowing the spindle to affect chromosome orientation and position. The molecular mechanisms by which this microtubule-based machine can accomplish chromosome organization and segregation are the subjects of my next two lectures.
The problems of segregating DNA
In spite of this special, chromosome-moving machine, the problem of chromosome segregation is difficult because each chromosome is made of one long piece of DNA. The chromosomes in our own cells are millimeters in length, i.e., thousands of times longer than the diameter of a human cell nucleus. Thus, the DNA of each chromosome must be condensed many fold to make an object that is small enough for the cell to move it around in an organized fashion. The “condensation” of each chromosome is achieved in part by wrapping the DNA around small clusters of proteins called “histones” to make “nucleosomes”, in part by packing the nucleosomes into helical fibers, in part by folding those fibers into loops, and in part by wrapping those looped fibers into yet another set of helices. This hierarchy of condensation shortens the DNA of each chromosome the necessary thousands of fold, making an object that is short enough to fit easily into a nucleus and thick enough to be seen in the light microscope.
DNA must be extended during interphase, so it can serve as a template for both RNA synthesis and DNA replication. As DNA replicates, the two identical double-stranded molecules that are produced become linked together by “cohesins”, so the two copies will be connected as they go into mitosis. These identical pieces of DNA are called “sisters”, and each one is commonly called a “chromatid”, rather than a chromosome. Every chromosome condenses in preparation for cell division during the stage of the cell cycle called “prophase”. Because S-phase precedes prophase, and because cohesins bind sister chromatids together, each chromosome is a double structure as it condenses for cell division.
Once condensation is far enough along, the mitotic spindle forms. It interacts with the now almost fully condensed chromosomes and organizes them into the 2-fold symmetric structure seen in “metaphase”, the stage just before the separation of the duplicated chromosomes.
The problems of segregating already duplicated chromosomes
The essential problem of mitosis is to get the sister chromatids of each duplicated chromosome attached to the two opposite ends of the spindle. This is accomplished in part because each chromatid contains one and only one site to which the fibers of the mitotic spindle can attach. This site, the “kinetochore”, is able to bind either the side or the tip of spindle microtubules. Stable connections between kinetochores and spindle fibers are made only when these links are under tension. This tension is produced largely at the kinetochore. It probably comes from both motor enzymes, like dynein, and from non-motor protein links that couple the kinetochore to spindle microtubules in such a way that microtubule depolymerization can exert force on the kinetochore. Once each chromosome is being pulled toward both of the spindle poles, the cell needs only separate the sister chromatids and allow them to respond to the forces acting on them. Then accurate separation of the duplicated chromosomes is achieved.
There are many different ways to look at a mitotic spindle
The process of mitosis can be followed with “phase” optics, in which chromosomes appear dark, but the spindle is essentially invisible. It can also be seen with “polarization optics”, in which the chromosomes are only ghosts, but the fibers of the spindle appear bright or dark. Fluorescence microscopy can be used in at least two ways to reveal aspects of spindle structure and function: by fixing cells to stabilize their structure, then staining particular spindle components with protein-specific antibodies tagged with a fluorescent dye (immunofluorescence), or by staining part(s) of the spindle with a fluorescent tag in vivo. The latter method can use both fluorescent dyes that bind to cell parts, like chromosomes, and mutant proteins that are made by coupling the gene that encodes a particular spindle component directly to the gene for a fluorescent protein, e.g., the green-fluorescent protein (GFP). If GFP is coupled to tubulin, the protein subunit of microtubules, the whole spindle is stained.
Spindles can be seen at higher resolution with the electron microscope, in which the fibers that attach to the chromosomes appear as bundles of microtubules. With this same instrument, the kinetochore often appears as a dark-staining layer on the underlying material of the condensed chromosome, the “chromatin”.
Protein localization by immunofluorescence and GFP-tagging has shown that kinetochores are complex assemblies of multiple proteins, probably around 100 different molecules. These many components give each kinetochore lots of useful properties in addition to microtubule binding: motor activity in two directions, enzyme activities that can help to depolymerize microtubules and protein kinase activities that can help to regulate the function of other proteins.
Each of these ways of looking at the mitotic spindle shows us a part of the full complexity of the machine that segregates the chromosomes.
The process of mitosis is somewhat different in different organisms
In yeast cells, the spindle is small; it forms inside the nucleus, whose membrane remains intact throughout mitosis. In mammals, vertebrates in general, and many higher plants, the spindle forms in the cytoplasm, but the nuclear envelope disassembles as the spindle forms, so the cytoplasmic microtubules gain access to the chromosomes as they condense in the nucleus. In some cells, like the flagellate, Barbulanympha, the nuclear envelope stays intact, the chromosomes condense in the nucleus, the spindle forms in the cytoplasm, and the two interact right through the nuclear envelope. Thus, there are numerous ways to solve the essential problems of mitosis. This kind of “biological variability” can make the study of mitosis complex, but it also give informative variation that can help the experimenter find a useful system for answering a particular question.
Some features of mitosis are almost universal
All spindles form with microtubules as their principal fibrous component. All spindles contain some microtubules that interact with kinetochores to organize the chromosomes and exert forces on them. All spindles form into a structure that is roughly 2-fold symmetric, and as the spindle acts on the duplicated chromosomes during anaphase, the structural symmetry plays out in the motion of the chromosomes to opposite ends of the cell, making it comparatively easy for a simple cleavage event to separate the mother cell into two similar and fully viable daughter cells.
Vocabulary: words whose meaning you should know.
DNA; chromosome; chromatin; replication; nucleosome; histone; condensation; cohesin; sister chromatids; interphase; S-phase; G1 and G2; differentiate; mitosis; microtubule; centrosome; kinetochore; prophase; metaphase; anaphase; nucleus; cytoplasm; nuclear envelope; phase optics; polarization optics; fluorescence microscopy; green fluorescent protein; electron microscopy; biological variability; 2-fold symmetry.