BIOL 129 Mitosis and the Cell Cycle
INTRODUCTION
Cell division is the process by which eukaryotic cells reproduce. The hallmark of
the process is the apportionment of an identical genome to each daughter cell. Division
of the nuclear contents is referred to as karyokinesis or, more commonly, mitosis.
Nuclear division is usually followed by cytoplasmic division or cytokinesis, the
apportionment of the cytosol and cytoplasmic organelles to the daughter cells as cell
boundaries are formed. In this project, you will study mitosis in root tip cells of the broad
bean, Vicia faba. It is a convenient and very popular system for studying mitosis because
the chromosomes are very large, the diploid number is relatively low (2n = 12), and cell
material is easily prepared. You will make squash preparations of the root tip cells and
identify all the stages of mitosis.
The Cell Cycle
In the life cycle of a eukaryotic cell, mitosis is part of a larger program, the cell
cycle. Actively dividing cells, such as in meristematic tissues of plants or in embryonic
tissues, are alternately in mitosis and in interphase. It is during interphase that cell growth
and DNA occur, the latter during the S period. There are two other periods in interphase,
G1 (1st gap), which precedes DNA synthesis, and G2 (2nd gap), which follows it. A cell in
G1 is considered to have the 2C level of DNA. During the S period, the amount of DNA
doubles to the 4C level and remains there until the end of division. After cytokinesis,
each daughter cell ends up with the 2C level of DNA. A mature eukaryotic cell that is
destined not to undergo further division is usually arrested prior to DNA syntheses, G1.
There is considerable variation both in the relative and actual duration of each stage
depending on the organism, cell type, and environmental conditions. In meristematic
cells of V. faba root tips, the cell cycle lasts about 19 hours. The specific durations of G1,
S, G2, and mitosis have been determined from an autoradiographic analysis.
Mitotic Stages
In the interphase nucleus, the chromosomal material is dispersed as a rather
homogeneous mass of fibers, referred to as chromatin. There may be several clumps of
condensed, deeply stained heterochromatin, and there is always at least one nucleolus.
In your preparations, nucleoli will appear as large, lightly stained, round or oval structures
within the nucleus. The stages of mitosis are prophase, metaphase, anaphase, and
telophase. While each mitotic stage is distinct, one stage does flow into the next so that
the adjectives early and late are often appropriate.
Prophase: Prophase begins when the individual chromosomes can first be discerned.
Though the chromosomes are long and thin, the double nature of each is apparent. The
two subunits, termed chromatids, are joined at a narrow chromosomal region known as
the centromere or primary constriction. Throughout prophase, the chromosomes
condense and shorten. The spindle assembles in late prophase, and the chromosomes
migrate toward a median position between the poles, a process termed congression.
These movements are effected by spindle fibers extending from the poles to the
chromosomes. The spindle fibers, which are composed of microtubules, terminate within
the kinetochore, a plate-like structure at the centromeric region of each chromatid. Other
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spindle fibers extend from pole to pole. The other event that occurs at late prophase is
the disassembly of the nucleoli and nuclear envelope.
Metaphase: At metaphase, the fully condensed chromosomes are lined up on the
metaphase plate, a plane that lies midway between the poles. Although all centromeres
lie on the metaphase plate, the chromosome arms are still quite long and extend laterally
to either side of the plate. Each metaphase chromosome has a characteristic
morphology determined by its size and centromere location. Metacentric chromosomes
have a median centromere and two arms of equal length. In acrocentrics, the centromere
is very close to one end, while in telocentrics it is at the tip. In V. faba there are 5 pairs of
acrocentric chromosomes and one pair of metacentric chromosomes; each metacentric is
about twice as long as each acrocentric. Another morphological feature apparent in
certain chromosomes is the secondary constriction. The secondary constriction is the
very thin, lightly stained chromosomal region where a nucleolus forms at telophase. The
chromosomal region distal to the secondary constriction is termed a satellite. In V. faba
there is a secondary constriction in one pair of chromosomes.
Anaphase: Anaphase is the climax of mitosis. The two chromatids part at the
centromere and move to opposite poles, and, in addition, the poles move farther apart.
The mechanism of anaphase movement is not fully understood but may involve spindle
microtubules sliding past each other. The shape of the anaphase chromosome depends
on the location of the centromere. The centromere leads the way to the pole so that a
metacentric chromosome is V-shaped, a submetacentric J-shaped, and a telocentric
rod-shaped. At late anaphase, the first sign of cytokinesis occurs with the formation of
the phragmoplast. The phragmoplast is a collection of dense material in small Golgi
vesicles associated with spindle fibers at the equatorial region.
Telophase: Telophase commences when the two daughter sets of chromosomes reach
the poles. Each daughter cell receives the same number of chromosomes as had been
present in the parent cell, though each chromosome now has only one subunit. There is
a general reversal of events that characterize prophase. The telophase chromosomes
decondense and become "fuzzy," the spindle fibers disassemble, and the nucleoli and
nuclear envelope reassemble, the latter from membranes of the endoplasmic reticulum.
Cytokinesis proceeds with the accumulation and fusion of vesicles at the equatorial
region. The fused membranes of the vesicles produce continuous plasma membranes
for the adjacent daughter cells, and the contents of the vesicles form the cell plate, which
eventually develops into the primary cell wall.
Mitotic Index
In any population of mitotically active cells, only some of the cells are in mitosis at
any one time. The percentage of dividing cells is defined as the mitotic index. The
approximate duration of mitosis can be obtained simply by multiplying the mitotic index by
the total duration of the cell cycle. You will determine both the mitotic index and the
duration of mitosis in V. faba root tips. For such an analysis, one should examine a cell
population that is mitotically active. Since not all cells in the root apical meristem are
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mitotically active, the values you obtain for the mitotic index and the duration of mitosis
will be underestimates. Having an estimate for the duration of mitosis, you will then
obtain estimates for the durations of the mitotic stages.
Root Tip Preparation
The beans were germinated and grown for one week under a lamp. They were
uprooted and briefly rinsed in water. 5 mm terminal tips of secondary (lateral) roots were
cut with a razor blade and then placed in freshly prepared Carnoy's fixative (absolute
ethanol:glacial acetic acid, 3:1). The tips were fixed for 24 hrs, transferred to 70% ethanol
and stored in a refrigerator at 4-5o C.
Feulgen Squash
The stages of mitosis are generally studied in squash preparations. Unlike
sectioned material, where only a part of the cell may be present, each cell in a squash
preparation contains all of the chromosomes. In addition, the squashing flattens the cells
so that the chromosomes are more dispersed and are often in the same focal plane. You
will start with fixed root tips that are ready for staining of the DNA by Schiff's reagent.
Other cell structures, like the spindle, either do not stain with Schiff's reagent or have
been destroyed by fixative or acid pretreatment. The HCL hydrolysis, which is part of the
Feulgen reaction, also macerates or softens the plant fibers so cells squash flatter. There
is no bisulfite rinse, as there is no need to verify the presence of DNA. After staining the
intact root tip, transfer it to a slide for squashing. The squash is done in a drop of 45%
acetic acid, which further softens the tissue. Cells are squashed by thumb pressure on
the coverslip.
PROCEDURES
Feulgen Squash
1. Rinse the fixed root tips briefly in distilled water and then place in a beaker of 1 N HCl
kept at 60oC for 10 min in a water bath.
2. Rinse the root tips briefly in distilled water and then place in a vial of Schiff's reagent,
stoppered and kept in the dark, for 30 min.
3. Rinse the root tips in several changes of distilled water. The intensely stained distal
tips contain the smaller, actively dividing cells.
4. Drain a root tip by touching it to a paper towel. Then, very gently rub the extreme tip
of the root on the towel to remove the root cap.
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5. Immediately place the tip on a labeled, alcohol-cleaned slide with a drop of acetic acid
and, with a sharp, single-edge razor blade, cut off and discard all but the terminal 1 mm,
which contains the dividing cells. Allow the tissue to remain the acetic acid for about 2
min.
6. Tap the root tissue with the flat end of a glass rod to produce a homogeneous
suspension in the drop of acetic acid. Gently lower a coverslip on the preparation.
7. Before squashing, remove the excess liquid as follows. Place a piece of paper towel
at one edge of the coverslip to draw out the excess liquid. Be very careful not move the
coverslip laterally, as this will cause the cells to fold over on themselves, ruining the
preparation.
8. Place a piece of paper towel over the coverslip and squash the preparation with the
thumb. Use a rolling motion so that the liquid is pressed to the edges where the towel
can absorb it. The pressure should be increased in successive rounds of squashing and
not applied all at once. Again, do not move the coverslip laterally.
9. Seal edges of the coverslip with thin layer of Vaseline applied with a toothpick.
Observations
1. Examine the preparation with the low power (10X) and high-dry (40X) objectives.
Identify and draw a cell at interphase, prophase, metaphase, anaphase, and telophase.
2. Examine several interphase nuclei, noting the prominent nucleoli. How many nucleoli
are there per nucleus?
3. Count 200 cells and determine the number of cells in the phases of mitosis and
interphase. To do this, move the slide to a position with nicely-squashed cells. Count the
number of cells in each mitotic phase and those in interphase. Then move the slide to a
new position and repeat the procedure until the total number of cells counted is 200. This
count represents a random sample of the entire population of cells, so do not search the
slide for a specific mitotic phase. Just move the slide and count the cells in view.
4. Total the number of cells in mitosis and interphase for the class. Using the combined
class data calculate the mitotic index.
5. From the value of the mitotic index and the known duration of the cell cycle in V. faba
(19 hr), estimate the duration of mitosis.
6. Determine the relative frequency of each mitotic stage. A combined figure should be
used for anaphase and telophase, since late anaphase and early telophase are difficult to
distinguish. Using the combined class data, calculate the durations of these stages.