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PLNT 3140 Introductory Cytogenetics - 2023

Making Sense of the Cell Cycle

Learning Objectives

1. The cell cycle separates gene expression, DNA replication and cell division into discrete phases. Be able to describe the main events in G1, S, G2 and M.
2. The cell spends most of its time in interphase, during which chromosomes are uncoiled, allowing gene expression to occur. Know the main components and functions of the interphase nucleus.
3. Be able to describe the main events in mitosis, and be able to identify which stage of mitosis a particular cell is in:

a. Prophase - condensation of chromosomes; disassembly of nuclear envelope into vesicles, attachment of spindle fibers.
b. Metaphase - Centrosomes pull spindle fibers taut, causing chromosomes to migrate to center of cell.
c. Anaphase - poleward movement of chromosomes through shortening of kinetochore spindles, after which poles are pushed apart by polar spindles.
d. Telophase - laminar vesicles reassemble into nuclear envelope; chromosomes decondense; cytokinesis
4. Be able to describe the functions of  polar and kinetochore spindle fibers.  What evidence is there that the anaphase migration of chromosomes is driven by depolymerization of kinetochore spindle fibers?
5. Be able to describe the nuclear envelope cycle.

The cell cycle is defined with respect to DNA replication

In the eukaryotic cell, the cell cycle divides the activities of gene expression, chromosome replication and cell division. Under the microscope, somatic cells seem to spend most of their time doing nothing. These long periods of inactivity are punctuated by relatively short periods where DNA synthesis and mitosis take place.

Idealized cell cycle, giving roughly proportional times. The times of cell cycle phases will differ with species and cell type.
Typical Eukaryotic Cell Cycle


The following experiment defined the phases of the eukaryotic cell cycle:

Cells in culture can be made to grow synchronously, which means that all the cells divide at the same time. To follow events in the cell cycle, you can feed the cells with radioactive nucleotides at different times, and measure the radioactivity remaining in cells after washing. Incorporation of radioactive nucleotides indicates that DNA synthesis was occurring at the time of sampling.

There is only a narrow time slot in which cells take up tritiated thymidine. This is the S phase, for DNA synthesis.

The phases of the cell cycle are organized by the two major cell changes: DNA replication and cell division. The other two phases are "gap" phases between those two activities.

1) G1 ("gap in DNA synthesis") - most  GENE EXPRESSION occurs here. In non-dividing or terminally differentiated cells, G1 persists indefinitely, and is referred to as G 0. Chromatin is dispersed in nucleus.
2) S - (DNA synthesis); The cell requires a discrete signal to begin a round of DNA replication. eg. if you fuse a cell in S phase with a cell in G 1, the nucleus from G 1 will begin DNA synthesis.
3) G2 ("second gap in DNA synthesis") The nucleus is reorganizing in preparation for mitosis. Some chromatin condensation occurs here, but not enough to be visible under light microscopy.
4) M - ("mitosis or meiosis") - chromosomes partitioned between two daughter cells.


Eukaryotic cells spend most of their time in interphase

Interphase, which includes G1 or G0, S and G2, is where the cell spends most of its time. Interphase is where most gene expression occurs. Put another way, most of what the cell does, other than dividing, occurs during interphase.


The nucleus in interphase organizes cell activities with respect to gene expression

Interphase, which includes G1 or G0, S and G2, is where the cell spends most of its time. Interphase is where most gene expression occurs. Put another way, most of what the cell does, other than dividing, occurs during interphase.

The purpose of the nucleus is to carry out the processes of gene expression and DNA replication. Inside the nucleus lies the chromatin, which refers to DNA complexed with different proteins. The interior of the nucleus is called the nuclear matrix, and the nucleus is enclosed by a nuclear envelope.

Nuclear pore complexes regulate molecular traffic into and out of the nucleus.

The envelope is actually two membranes: the inner membrane has attachment sites for chromosomes, and the outer membrane helps define the shape of the nucleus. The space between these two membranes is called the perinuclear space.

The traffic of different molecules in and out of the nucleus is controlled by nuclear pores which are protein complexes of their own.

VIDEO: Into the Nucleus - animation of import into nucleus via nuclear pore http://youtu.be/UyhqLpjicZg The nuclear pore complex consists of stringlike fibrils that project into the cytoplasm, a central meshwork, and a nuclear basket on the inside.


Displayed from http://pubs.rsc.org/services/images/RSCpubs.ePlatform

Generally, molecules can go in or out of the nucleus - not both. RNA molecules made inside the nucleus are transported out for translation by ribosomes. Proteins made outside the nucleus are transported in for use inside.

What are some of the proteins that get transported into the nucleus?



The nucleus may have evolved from the endoplasmic reticulum

The nucleus is the main difference between prokaryotic (pre-nucleus) and eukaryotic (true nucleus) cells. So how did this nucleus come to be? If we look closer at the image from above, we can see that the nuclear envelope seems to be continuous with the rough endoplasmic reticulum (RER).

If we consider the nuclear envelope as a differentiated form of the RER, there would have been some clear advantages to cells which formed a nucleus. RNA transcripts that exit the nucleus come into immediate contact with ribosomes for translation. Proteins translated that need to go back to the nucleus don't have far to go. In this model, the formation of a nucleus would have been preceded by the formation of a rough (ribosome-prevalent) endoplasmic reticulum in the first place.


MITOSIS

The distinction between mitosis and meiosis

Mitosis - nuclear division leading to  equal sets of chromosomes  in the daughter nuclei. Characteristic of somatic cells.
Meiosis - nuclear division leading to a  reduction in chromosome number of the parent cell to produce haploid gametes.

The mechanism of mitosis

During interphase, the chromosomes need to be loose so that gene expression can take place. But when it comes time for the cell to divide, loosely coiled chromosome spaghetti isn't easy to separate. So, the chromosomes have to get a lot smaller and denser to divide in an orderly fashion.

When the chromosomes have coiled enough that they retain stains (and we can see them), we call that prophase. The nuclear envelope is also dissasembled during prophase. In metaphase, we see the chromosomes attached to spindle fibres and lined up at the centre of the cell. During anaphase, the "tug-of-war" between the chromosomes and centrosomes resolves, and we see the chromosomes being dragged to opposite ends of the cell. Finally, in telophase, the nuclear envelope reassembles, the chromosomes decondense, and the nucleus reorganizes itself, allowing normal cellular functions to resume.

Interphase

It is important to remember that interphase is defined by what we can see under the microscope with conventional staining techniques.  While it may look like nothing is happening, we have already seen that the term "Interphase" includes G1, S and G2 phases of the cell cycle.
 Onion root tip cells in interphase, stained with aceto carmine. Nuclei stain red, nucleoli appear white.
Image from Yaping Wang and Brian Fristensky, University of Manitoba.


Organization of spindle fibers prior to mitosis

The role of centrosomes in the cell cycle are illustrated in the  figure. (from http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.figgrp.2472 ) Prior to the visible onset of prophase, the following steps occur:

Centrosomes are the site of spindle fiber organization, and thus provide a polarity to the dividing cell.  They  also serve as "basal bodies" in cells with flagellae.

From https://en.wikipedia.org/wiki/Centrosome

Spindle fibers are microtubules which originate at the centrosomes. Microtubules are tubes formed from polymers of α- and β-tubulin heterodimers. As illustrated above, bundles of microtubules also form the centrosomes. Centrosomes are also referred to as "microtubule organizing centers" (MTOC).


Prophase

1. The initiation of chromatin condensation coincides with the disassembly of the nuclear membrane. 

Prophase in onion root tips with aceto-carmine staining. A - C show the progression of chromosome condensation. Note that as chromosomes condense, they become thicker and less diffuse.

Images from Yaping Wang and Brian Fristensky, University of Manitoba.
 A
B

 C

There is an observed increase in the density of chromosomes during prophase, as the cell prepares to divide its contents. Meanwhile, the nuclear envelope is dissolving to allow the later events of mitosis to take place unhindered.


2.  Switch in microtubule stability accompanies formation of spindle fibers.

Spindle fibers extend randomly in all directions. The length of the fibers varies due to  polymerization and depolymerization of the microtubule from tubulin dimers. Polymerization occurs at both ends, but is relatively slow at the (-) end, which is anchored at the centrosome. The (+) end grows more rapidly, as it extends into the cytoplasm. However, when a microtubule encounters the kinetochore at the centromere of a chromosome, it's (+) end is  stabilized by the kinetochore. The kinetochore is a protein matrix, consisting of proteins that attach to centromeric DNA sequences (inner layer) and proteins that attach to the spindle fibers (outer layer).

The kinetochore is a protein/DNA complex which forms at the centromere. It is the site of attachment of kinetochore microtubules to the mitotic chromosome.
Fluroescently-tagged spindle fibers are shown in green. DNA in chromosomes is counterstained in blue flurorscence.

http://en.wikipedia.org/wiki/Image:Mitotic_spindle_color_micrograph.gif


3. By end of prophase, sister chromatids have separated but remain joined at centromeres.

Despite the fact that DNA replication occurs prior to prophase, the two sister chromatids cannot be distinguished microscopically during early prophase. This is referred to as relational coiling.
As the chromosomes condense during prophase, the relational coils are unwind, and each chromatid coils tightly with itself.  Relational coiling disappears as prophase continues and the chromatids disengage to lie side by side.


Displayed from
https://www.nature.com/scitable/content/18070/10.1038_nrm2310-f2_large_2.jpg

In summary, prophase is characterized by:

Metaphase

In metaphase the chromosomes are fully condensed and arranged in a plane equidistant from the poles of the spindle. This is the highest level of coiling, and the chromosomes are shorter and thicker than any other stage and therefore ideal for cytogenetic study. There is no longer much relational coiling present, and the chromatids lie side by side, not twisted around each other.

Experimental evidence indicates that centrosomes appear to pull the spindle fibers taut, in a kind of "tug of war". Thus, the chromosomes migrate toward equator of the cell and become aligned.
VIDEO (12.7 Mb) PLNT3140-CongressionOfChromosomes.webm

Metaphase in onion root tips.
Image from Yaping Wang and Brian Fristensky, University of Manitoba.

In summary, metaphase is characterized by:


Anaphase

Anaphase is the shortest phase of mitosis. The beginning of anaphase is observed microscopically when the synapsed sister-chromatids separate simultaneously, and begin migrating to the opposite cellular poles. During anaphase, longer chromosomes often appear V-shaped or J-shaped.

Early anaphase
 Late Anaphase
Images from Yaping Wang and Brian Fristensky, University of Manitoba.

kinetochore fibers - spindles attached to kinetochore.

polar fibers - spindles interdigitated with spindles from opposite poles

1. Poleward movement of chromosomes (anaphase A) is powered by microtubule depolymerization.

Begins with a splitting of the kinetochore, releasing the tension and allowing chromosomes to "dangle" at the ends of their respective spindles.  Depolymerazition of microtubules at the kinetochores causes sister chromatids separate into independent chromosomes.  Depolymerization provides energy for chromosome movement.

DEMONSTRATION THAT CHROMOSOMES MOVE POLEWARD ALONG STATIONARY KINETOCHORE MICROTUBULES [Adapted from G. J. Gorbsky, P. J. Sammak, and G. Borisy, 1987, J. Cell Biol. 104:9; and G. J. Gorbsky, P. J. Sammak, and G. Borisy, 1988, J. Cell Biol. 106:1185.]
The experiment shown above verifies this model. At top, cells have been labeled with fluorescently-tagged tubulin proteins, so that microtubules (eg. spindle fibers) fluoresce red in UV microscopy. A laser is used to bleach part of the spindle in metaphase cells, so that the bleached spot will not fluoresce.  During anaphase, the distance from the chromosomes to the bleached spots decreases, while the distance from the centrioles to the bleached spots remains constant. This suggests that the kinetochore spindle fibers are shortening due to depolymerization at the kinetochore.

2. Separation of the poles (anaphase B) involves sliding of adjacent microtubules, requiring ATP

A second set of spindle fibers, called "polar fibers", are not attached to the kinetochore. Rather they are interdigitated with each other. That is, one polar fiber from one centrosome polymerizes until it overlaps a fiber from the opposite pole.
At the end of anaphase, the spindle fibres converge at the pole and the chromosomes are jammed together. Polymerization of + ends of polar fibers push the poles apart, bring the chromosomes with them.

In summary, anaphase is characterized by:

Telophase and Cytokinesis

Telophase starts when chromosomes reach opposite poles and form a dense chromatid ball. It is the termination of mitosis and the most difficult stage to study cytogenetically because the chromsomes are crammed into a small space, which will reconstitute into an interphase nucleus.

During telophase, the nuclear envelope is reconstituted from vesicles leftover from the parent nucleus, and chromsomes begin to decondense. Nucleoli, the nuclear matrix and the nuclear membrane reform, and the spindle fibers disassemble. Telophase restores the daughter cells to the interphase state.


What happens to the nuclear envelope during mitosis?

At the onset of mitosis, the nuclear envelope dissociates into vesicles, storing the envelope materials for later use during telophase, when the vesicles are reconstituted into a new nuclear membrane.

The re-use of envelope materials minimizes the amount of these materials that need to be synthesized from scratch each cell cycle.
Displayed from http://upload.wikimedia.org/wikipedia/commons/8/8a/Nuclear_envelope_breakdown_and_reassembly_in_mitosis.jpg

Telophase restores the daughter cells to the interphase state. Envelope vesicles first enclose each individual chromosome, after which vesicles fuse to form a single genetically complete nucleus at each pole. Nucleoli and the nuclear membrane reforms and the spindle fibres disappear. Chromatin decondenses, and the nuclear matrix re-assembles.

Cytokinesis

The division of the cytoplasm and its organelles between daughter cells is called cytokinesis, which begins during late telophase at the equatorial plate. In plants, cytokinesis takes places by the formation of the cell plate. In animals, cytokinesis works by formation of a cleavage furrow, pinching off the cell much like a drawstring. Generally, cytokinesis divides the parent cell equally between the two daughter cells. Sometimes, no cytokinesis occurs after nuclear division, resulting in a binucleate cell. Additionally, division can be asymmetrical and give rise to daughter cells of different shapes and sizes.

In summary, telophase is characterized by:


Summary