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This is to certify that the

thesis entitled

A:

MITCTIC CYCLE I}? EISUB-‘I S'TI‘FUM

presented by

Lee Virn Leak

has been accepted towards fulfillment
of the requirements for

% degree in- BQVPHI

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Major professor
Date 2 z /7/6 2

  

LI BR A R Y
Michigan Static
University

 
    

 

[LN ELECT Cl-J IIICRCECCEIC JILCLYSIE. CF TE:
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THUCTU“? OF‘ JIEIIST,L-..’TIC CELLS gLTRL.

 

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AN ELECTRON MICROSCOPIC ANALYSIS OF THE FINE STRUCTURE
OF MERISTEMATIC CELLS DURING THE MITOTIC

CYCLE IN PISUM SATIVUM

BY

Lee Virn Leak

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1962

 

 

 

 

ABSTRACT

AN ELECTRON MICROSCOPIC ANALYSIS
OF THE FINE STRUCTURE OF MERISTEMATIC CELLS
DURING THE MITOTIC CYCLE IN
PISUM SATIVUM

by Lee Virn Leak

This study was undertaken to investigate fixatives for
electron microscopic studies that would reveal the fine
structure of the spindle element along with the preservation
of other cellular components. Observations revealed that
2, PbClZ,

chr207 and Cro3 in a 7%.to 10% formalin solution preserve

the fixatives containing metallic salts as CdCl

the fine structure of the spindle, however, the fine struc-
ture of the cytoplasmic organelles were often distorted. A
formalin and modified 0304 fixative gave detail spindle
structure along with the preservation of other cytoplasmic
components. By using the above fixatives the sequence of
spindle development could be studied in the meristematic
cells of giggm sativum during active mitosis.

The chromosomal and interzonal fibers emanate from the
chromosomes with the chromosomal fibers migrating toward

the polar regions and the interzonal fibers occupying the

mid-region of the cell. The continuous fibers extend from

 

 

 

 

Lee Virn Leak

one polar region to the other and are attached to the

chromosomes by fine fibrils.

The fibers are composed of fibrils with granules along
their length.

The phragmoplast is formed from the interzonal fibers
and is separated in two units by the cell plate which is
formed by a process of granular fusion or polymerization
across the mid—region of the cell.

The fibrillar orientation is disrupted when cells are
treated with a threshold concentration of colchicine. As
a result of this disruption the chromosomal fibers, inter-
zonal fibers, continuous fibers, phragmoplast, and cell
plate are not formed. Hence, cytokinesis does not occur,
and the resulting cell is either a polyploid, binucleate or
Inultinucleate one.

Cytoplasmic components migrate or are pushed by the
action of the spindle body (during mitosis) in equal pro—
;xzrtions to the ends of the cell for redistribution in the

daughter cells.

 

 

 

 

 

 

 

 

 

 

 

 

 

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to
his colleagues in the cytology group at Michigan State Uni-
versity for their aid during this investigation.

To Dr. G. B. Wilson, special thanks for the continued
guidance and inspiration that were always present throughout
the course of this study.

My sincere appreciation to Mr. P. G. Coleman for his
excellent photographic services. To Dr. J. A. Bergeron and
Mr. M. Gettner of Brookhaven National Laboratory, Upton,

N. Y. many thanks are given for teaching the principles of
electron microscopy as applied to the study of biological
systems.

Also sincere appreciation for the helpful suggestions
from Drs. J. R. Shaver, L. W. Mericle, E. Cantino and W. B.
Drew.

In addition acknowledgment is made to the Agricultural
JExperimental Station for financial support of the author
<iuring his graduate studies and for partial support of
research expenses. Thanks is offered to the Division of
JBiological Science of Michigan State University for partial

financial support.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . 3
.MATERIALS AND METHODS . . . . . . . . . . . . . . l3
OBSERVATIONS . . . . . . . . . . . . . . . . . . . 18
DISCUSSION . . . . . . . . . . . . . . . . . . . . 27
SUMMARY . . . . . . . . . . . . . . . . . . . . . 39
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . 41
EXPLANATION OF FIGURES . . . . . . . . . . . . . . 51

TEXT FIGURES . . . . . . . . . . . . . . . . . . . 53-129

iii

Figure

10.

ll.

12.

13.

14.

15.

16.

17.

18.

19.

LIST OF TEXT FIGURES

A lowépower image of interphase and prophase

Late prophase . . .
Interphase and late prophase

Diagram of a probable method of spindle
production . . . . . . . . . . . . . . . .

light micrograph of metaphase and telophase
Pro—metaphase

Chromosomal fibers of metaphase cell

Bundle of chromosomal fibers . . . . . . . .

Attachment of chromosomal fibers to

chromosome . . . . . . . . . . .
Continuous fibers . . . . . . . . . .
Metaphase plate . . . . . . . . . . . . . . .

Metaphase plate . . . . .
Metaphase plate . . . . . . . . . . . . .
Interphase cell showing cytoplasmic components

Interphase cell showing cytoplasmic components

Early anaphase .

Early anaphase . . . . . . .
Mid—anaphase . . . . . . . . . . .
Mid-anaphase . . . . . . . . . .

iv

Page

53

55

57

59
61
63
65

67

69
71
73
75
77
79
81
83
85
87

89

 

 

1:» “WM 5': '

 

 

Figure Page

20. Mid-anaphase . . . . . . . . . . . . . . . . 91
21. Anaphase . . . . . . . . . . . . . . . . . . 93
22. Late anaphase . . . . . . . . . . . . . . . 95
23. Enlarged interzonal fibers . . . . . . . . . 97
24. Late anaphase . . . . . . . . . . . . . . . 99
25. Anaphase . . . . . . . . . . . . . . . . . . 101
26. Anaphase . . . . . . . . . . . . . . . . . . 103
27. Early telophase . . . . . . . . . . . . . . 105
28. Phragmoplast . . . . . . . . . . . . . . . . 107
29. Phragmoplast . . . . . . . . . . . . . . . . 109
30. Developing cell plate . . . . . . . . . . . 111
31. Developing cell plate . . . . . . . . . . . 113
32. Developing cell plate . . . . . . . . . . . 115
33. Cell plate . . . . . . . . . . . . . . . . . 117
34. Cell plate . . . . . . . . . . . . . . . . . 119
35. Reorganization of nuclear envelope . . . . . 121
36. Daughter nuclei . . . . . . . . . . . . . . 123
37. "Scatter" . . . . . . . . . . . . . . . . . 125
38. "Clump" . . . . . . . . . . . . . . . . . . 127
.39. Tetraploid interphase . . . . . . . . . . . 129

INTRODUCTION

In his never-ending search for an explanation of the
many complex phenomena occurring during embryogenesis and
continuing throughout the life span of the living organism,
the biologist is constantly prompted to investigate the
numerous processes occurring within the living organism
under diverse experimental conditions. Such investigations
led Robert HOoke to the discovery of the cell, which was
later proposed by M. Schleiden, the botanist, and T.
Schwann, the zoologist, to be a basic unit of plants and
animals. Later this theory was confirmed by R. Virchow as
he showed that all cells come from pre-existing cells
(OMNIS CELLULA E CELLULA).

In his attempt to resolve the complex phenomena of the
living organism, the biologist must continue to probe the
Inicro and ultra-fine structures of the cell as well as to
seek correlation with function. The application of the
electron microscope in the area of biological research has
Inade it possible to begin bridging the chasm between the
Inicron and angstrom levels.

The mechanism of cell division has been a subject of
constant analysis since its early observation by Fleming,

(3. Hertwig, Van Beneden and others in the late 1800's. In

1

all higher plant and animal cells, in protozoa, in most
algae and fungi, cell replication involves the method of
mitosis. With the aid of phase microscopy and cine-
micrography, we are able to observe this dynamic process
in the living cell, especially by utilizing tissue culture
methods.

Mitosis is but one phase of the mitotic cycle that one
can readily identify by well—defined morphological features
as: (l) a condensation of genetic material in the form of
chromosomes, (2) nuclear disruption which involves the
breakdown of the nuclear membrane, (3) the separation and
movement of sister chromatids to opposite poles, and (4)
reconstitution of daughter nuclei. The second part of the
mitotic cycle has been termed the synthetic phase, in which
the synthesis of DNA, RNA and proteins occur.

The movement phenomena of mitosis have been analyzed by
numerous investigators, and the mechanisms explained by
various hypotheses, in which a great deal of importance is
placed on the spindle element and its relation to chromo-
some movement.

Early observers generally considered the spindle fibers
to be a fixation artifact. Hewever, as techniques in
histology and cytology were improved, these structures were

shown to be definite parts of the mitotic apparatus.

Recent investigations utilizing electron microscopy
have revealed that the spindle element is composed of a
definite fibrillar structure. Therefore, the present inves—
tigation was undertaken to determine: (1) the spindle

formation in the meristematic cells of Pisum sativum,

 

(2) its fibrillar nature as revealed by electron micro-
scopic studies, (3) cell plate formation, (4) cytokinesis,
and (5) the effect of colchicine on the fibrillar structure

of the spindle fiber and formation of the cell plate.

LITERATURE REVIEW

The early information on the mitotic spindle as a
fibrous structure was obtained from observations of fixed
cells. Observations of living, untreated dividing cells,
however, frequently revealed the mitotic spindle to be
optically homogeneous (Cooper, 1941). Chambers (1924) was
unable to demonstrate fibers in the spindle by using
microdissection techniques. Since the fibrous structure
was observed mainly in the fixed cell, this led to the
belief that the spindle fibers have no morphological reality
as such. Because of this apparent lack of structure in the
living cell, the spindle was regarded as being the result
of faulty fixation (Martens, 1929; Robyns, 1929; and Bleier,
1931a and b). Lewis (1923) was able to show the spindle
fibers in chick tissue culture cells only when the medium
'was brought down to pH 4.6. Because of the association
‘with pH change, she concluded that the fibers were arti—
facts. During this time the use of vital stains had added
‘very little to the demonstration of the spindle in the
living cell, although these dyes stained the nuclear com-
'ponents and some cytoplasmic organelles, there was always

'the possibility of damage to the living cell (Ries, 1938).

Reality of the Spindle: NUmerous experiments by various
investigators have been carried out in an effort to settle
the question of the reality of the spindle element. Balar
(1929a and b), Schrader (1934) and Schmidt (1936a, b and c)
showed optical anisotropy in the spindle of living sea
urchin eggs. Cooper's (1941) observations of dividing
blastomeres of the living, untreated eggs of the mite,
Pediculopsis qraminum, with the light microscope, strongly
suggested that fibrous structures comprise the spindle
elements in these dividing cells. Carlson (1952), contrary
to the results of Chambers' investigations, showed that the
metaphase spindle in the living cells of Chortophage,
including chromosomes and asters, constitutes a physical
entity that can be moved about with the aid of a micro-
needle. In the spermatocytes of some Coccidae, Hughes-
Schrader and Ris (1941) were able to observe the spindle
as a unit before the nuclear membrane had broken down.

In testing for the reality of the spindle fibers, Lillie
(1909), Morgan (1910), Spooner (1911) and Andrews (1915)
subjected living cells to centrifugation and found that the
fibers could be bent or torn. Later studies of this nature
were conducted by Schrader (1934), Beams and King (1936)

and Shimamura (1940) with similar results.

In addition to the demonstrations of Cooper (1941) and
Cleveland (1938a and b) of the chromosomal fibers in the
living spindle with the light microscope, further evidence
for the reality of the spindle was provided by Schmidt (1936a
and b, 1939) with the aid of polarized light. Under such
optical conditions, the living spindle shows positive
birefringence in its long axis. Since protein molecules
are known to behave in this fashion, it was concluded that
there existed a longitudinal structure made up of protein
molecules with parallel orientation. Confirmation of these
conclusions may be found in the studies of Runnstrom (1936),
Pfeiffer (1940), Hughes and Swann (1948) and Inoue (1953).
X-ray diffraction experiments by Schmitt (1940) also further
substantiate these conclusions. These observations together
with the demonstration of birefringence in fixed spindles
identical to that produced by the living spindle, led
Schmitt to the belief that fixation induces no fundamental
changes in it, and that the permanent preparations probably
give a true picture of the actual conditions.

With his modification of the polarizing microscope,
Inoue (1951, 1953) confirmed the evidence for birefringence

in the spindle and demonstrated a definite fibrous structure

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- m ..a_ .
m .

in the living mitotic spindle similar to that which had
already been observed in the fixed cell.

The observations of Bajer (1951, 1957), utilizing cine-
micrographic methods, and those of Taylor (1959) also pro-
vide evidence that the spindle is an organized fibrillar
region.

Origin of the Spindle Element: The studies of Bleier
(1930a and b), Koerperich (1930), Wada (1935) and Becker
(1938) suggested that the spindle fibers of the metaphase
configuration are located in a substance that is quite
distinct from the cytoplasm. These investigators main—
tained that the spindle is probably derived entirely or in
a greater part from the extra-chromosomal content of the
prophase nucleus. Wada (1941) concluded that, in the case
of plants, the spindle body appears to be entirely intra-
nuclear in origin. On the other hand, observations of
.Mazia (1956) and Bajer (1957) suggested that the spindle
‘body is at the outset, of cytoplasmic origin. Bajer
:reported that the first sign of the spindle rays are those
.fibers that extend from the centrioles to the equatorial
plane (Painter, 1916).

The interzonal connections extending only between the
separating chromatids make up the interzonal fibers (Schmidt,

1939),

In the plant cell the interzonal region is linked with
the development of the phragmoplast and the subsequent for-
mation of cell plate or cell wall that is associated with
cytokinesis.

Esau (1960) defines the phragmoplast as a fibrous
structure that appears at telophase of a mitosis between
the two daughter nuclei, and plays a role in the formation
of the initial partition, dividing the mother cell in two
units. It is a barrel shaped region between the daughter
nuclei which becomes flattened and ring-like as cytokinesis
begins.

Cytokinesis: The division or separation of the extra-
nuclear material into two units is referred to as cyto-
kinesis or cell division. In plant cells the new cell
wall is also formed at this time. In somatic divisions,
which characterize growth as a result of meristematic
mitotic activity, there is a close correlation between
nuclear and cellular division. However, these operations
are separate in the formation of pollen and endosperm in
many angiosperms, and in the development of the female
gametophyte and the proembryo in gymnosperms (Sharp, 1934).

The inception of cytokinesis is marked by the appearance

of a membrane-like line between the daughter nuclei. This

line which arises in the equatorial plane of the fibrous
spindle (the phragmoplast) is referred to as the cell
plate (Esau, 1953).

According to Esau (1953), in some cases during the
early prophase of nuclear division, before the inception
of cytokinesis, a cytoplasmic plate, the phragmosome, is
formed across the cell in the plane of the division. It
is derived from strands of parietal cytoplasnu 'and thus
forms a medium in which the phragmoplast and the cell
plate will develop.

In his studies, Yasui (1939) observed the appearance
of fine granules at the equatorial region of the phragmo—
plast, noting that they increased in number and fused
together, resulting in the formation of the cell plate.
Yasui divided the process of cytokinesis into two phases:
(1), the bipartition of the cytoplasm by the lateral growth
of the phragmoplast, and (2) the bipartition of the
phragmoplast by the formation of the cell plate in its
equatorial plane.

More recently, Porter and Machado (1960) observed the
occurrence of numerous circular and oval bodies in the
region of the developing cell plate that appeared to be

associated with the phragmoplast, and to represent an early

 

 

 

10

stage in the formation of the cell plate. They disappeared
with the completion of separation of the daughter cells, as
though they were used up in the process of formation of the
middle lamella. Because of their association with the
development of the cell plate, Porter and Machado referred
to these bodies as phragmosomes.

The Structure of the Spindle Element: Studies of the
spindle element in the living or fixed cell with the light
microscope, the polarizing microscope, and by x-ray dif-
fraction methods give evidence that its structure is
fibrillar in nature (Inoue, 1953; Schmitt, 1940).

Recent investigations with the electron microscope have
revealed both the astral and spindle fibers as being com-
posed of fine fibrils 150-200 A thick and several microns

in length (Beams t a1,, 1950, Amano, 1957; Bernhard and

deHarven, 1958; Bessis §t_§1,, 1958). Sedar and Wilson
(1951) and Sato (1958, 1959 and 1960) showed that the
spindle fiber is composed of submicroscopic fibril units.
Sato's studies revealed the fine structure of the phragmo-
plast as consisting of a large number of parallel fibrils
lying vertical to the equatorial plane, and he also noted

that a fluid substance of low electron density fills up

the interfibrillar space.

ll

Spindle Disruption by Colchicine Treatment: Chemicals
that inhibit mitosis in metaphase have been referred to as
spindle poisons or "C-Mitotic" agents. Cytological studies
by Ludford (1936), Nebel and Ruttle (1938), Eigsti (1938)
and Levan (1938) indicated that colchicine stops mitosis
in the living cell, while those of Hadder and Wilson (1957)
and Molé-Bajer (1958) showed that colchicine disrupts
specific phases of the mitotic cycle. Treatment of the
isolated mitotic apparatus of sea urchin eggs with col-
chicine produced an amorphous gel, indicating that col-
chicine could affect the secondary bonding assumed to
contribute to the integrity of the spindle structure
(Mazia, 1956). It seems that in colchicine-treated eggs
(sea urchin), the protein fibrils that constitute the
normal spindle form an irregular network instead of lying
parallel. It is probable that the protein molecules which
build up the spindle fibers are altered in their composi—
tion or structure by colchicine. Thus they would still
form protein fibers through oxidation of -SH- groups but
these fibers would be incapable of parallel orientation by
hydrogen bonding (Mazia, 1956).

The observations of Inoue (1952) and Swann (1951) with

the polarizing microscope showed that the orientation of

.__ 0‘;

f" (1)..

 

 

 

 

 

12

the protein fibers is changed after colchicine treatment.
The inhibition of the development of the spindle in col-
chicine-treated cells is determined by the concentration of
the colchicine used and the duration of exposure. Gaulden
and Carlson (1951), Hadder and Wilson (1957) and Van't
Hof (1961) showed that the greater the concentration and
time of exposure, the greater will be the degree of dis-
organization of spindle or interference with its development.
By using short exposures with near threshold concen-
trations (2.5 X 10-4 M and 2.5 X 10-3 M) of colchicine,
Sedar and Wilson (1951) showed a progressive swelling and

solubilization of fibrous material as revealed by the

electron microscope.

MATE RIALS AND METHODS

The observations reported in this investigation were
made from the first 2-3 mm of the distal portion of the
meristematic region of the root tips of pea seedlings,

Pisum sativum var. Alaska, which were furnished by the
Ferry-Morse Seed Company.

The peas were soaked for six hours in distilled water
at 250 C, then rolled in paper toweling and placed verti-
cally in 250 ml beakers that were one-third filled with
distilled water. Wax paper was placed around the rolled
toweling to prevent the peas from drying out by evaporation
of the water. The peas were allowed to germinate in an
incubator at 250 C for 48 hours, at which time the primary
root had reached a length of 3-5 cms.

Seedlings with the desired root length (2.5 - 3.5 cms)
were suspended on waxed, one—quarter inch wire mesh over
250 m1 beakers which contained one-quarter strength Hoagland's
nutrient (balanced salt) solution. The one-quarter strength

Hoagland's solution consists of the following in grams per

liter:
Ca(NO3)2 -------------------- 0 0246
NH4NO3 -------------------- 0 033
M9304 7H20 --------------- 0 0462

l4

Seedlings to be used as controls were placed in the
nutrient solution and samples taken at zero hrs., 4 hrs.,
8 hrs., 12 hrs., and 24 hrs.

Seedlings to be treated were placed in beakers con—
taining the colchicine and the nutrient solution. After
treatment was completed, the seedlings with the wire mesh
were removed from the solution of colchicine, washed and
placed in a beaker containing only the nutrient solution.

The solution used had a pH range of 5.5 - 5.7. One M
HCl and a 3.57 M KOH solution were used when pH adjustments
were necessary.

The colchicine used in the experiment was purchased
from Light's Organic Chemical Ltd., and its structural

formula (Muldoon, 1950) has been determined to be:

CHNHCOCH

H
C-———-—CH
l 2
C
3
l l

     

.— ~
‘ -Y-fi .. v.

 

 

 

 

15

The colchicine solution was prepared shortly (5-10
minutes) before use in order to obviate chemical change.
Seedlings were treated for 30 minutes with a 4.5 x 10_4 M
concentration of colchicine.

Tips 1 to 2 mm in length were harvested with a sharp
razor blade, with some tips being split in half longitu—
dinally and immediately placed in a fixative.

Of the cellular components, the spindle element has
been the most difficult to demonstrate consistently while
still preserving other cellular organelles.

Presently, the electron microscopist does not have at
his disposal the ”Universal Fixative” which will preserve
simultaneously the chromosomes, spindle element, and the
major cytoplasmic organelles. In experimenting with a
range of fixatives it was found that the heavy metallic
salts as: CdCl , CoNO , CrSO , K Cr 0 PtCl , SnCl and

2 3 4 2 2 7' 6 4

HQCl gave detailed spindle and fair chromosome preserva—

2
tion while the cytoplasm and its components were highly
disrupted.

By using the following fixatives (A and B) in a 1:1

ratio the spindle element along with other cytoplasmic and

nuclear components were preserved:

 

‘ v w
,1,

 

 

 

Solut:

The ab
RIHOaglanc
Tm spindle
be demonstr
BY adding C
adjusting t
demonstrate;
fixed in bu;

Tips we:
PrepiOnic ac
MGht micros

FixatiOr

 

fixatiOn/ t?

l”creasing C

nitrate to c

.H ‘
met-1’1 m

l6

 

Solution A: 4% CrO3 ----------------- 10 ml
4% KZCrZO7 -------------- 10 ml
10% formaldehyde -------- 10 ml
1% NaCl ----------------- 10 ml
Saturated picric acid --— 5 ml

The above salts with the exception of NaCl were made up
in Hoagland's nutrient solution, and had a final pH of 3.5.

Solution B: 2% 0504 made up in the nutrient solution.

 

The spindle element along with cytoplasmic components could
be demonstrated when solution B was adjusted to pH 5 - 5.6.
By adding CdCl2 to solution A to make a 2% solution and
adjusting the pH to 5 - 5.6 the spindle could also be
demonstrated. Tips were also fixed in formalin and post
fixed in buffered 0504 at pH 7.2 and pH 5.6.

Tips were also fixed in an ethanol, formaldehyde and
propionic acid fixative (Leak and Wilson, 1960) for routine
light microscopic analysis.

Fixation time ranged from 15 minutes to 12 hours. After
fixation, the tips were washed, rapidly dehydrated in
increasing concentrations of ethanol with 0.01% uranyl
nitrate to cut down on local explosion. Tips were embedded

in normal butyl methacrylate or 90% butyl methacrylate and

10% methyl methacrylate and catalyst with 0.01% uranyl

 

 

nitrate

zation a

allowed

 

and then
Tips

in Epon 5
Sectic
Moran, 195
tome. Thi
Ones and e)

order to de

r0“ tip ( o

 

 

l7

nitrate added to reduce explosion due to uneven polymeri—
zation as recommended by Ward (1958). Polymerization was
allowed to occur in an oven starting at 370 C for 24 hours,
and then increased to 600 C for an additional 24 hours.

plus 030 and 030 were embedded

Tips fixed in KMnO 4, 4

4
in Epon 812 according to Luft (1959).

Sections were obtained with a diamond knife (Fernandez-
Moran, 1954, 1956) and glass knives on a Leitz ultra micro-
tome. Thick sections 5—10 microns were cut adjacent to thin
ones and examined with the phase contrast microscope in
order to determine the exact position of the cells in the
root tip (Ornstein and Pollister, 1952). Methacrylate
sections were spread by holding a wooden stick moistened
with xylene over the sections in the knife trough. Grey
(approximately 30-60 mu thick) and silver (approximately
60-90 mu thick) sections (Peachey, 1958) were placed on
copper grids covered with a parlodion film and coated with

a thin film of carbon (Pease, 1960). Sections were observed

with a Philips 100B electron microscope.

 

 

 

The 01
obtained f
sections f
sativum.

In the
cells of pl
material in
sones along
Prophase stg
nuclear divi
the Chromoso
filamentous
A Subsequent
the terminat

nuclear enve

 

 

OBSERVATIONS

The observations described in this investigation were
obtained from studies of numerous electron micrographs of
sections from the meristematic region of root tips of Pisum
sativum.

In the meristematic cells of Pisum, as in most mitotic
cells of plants and animals the condensation of the genetic
material in the form of filamentous or rope-like chromo-
somes along with a spherical or ovoid nucleolus marks the
prophase stage, i.e., the inception of active mitosis or
nuclear division (fig. 1). With the progression of prophase,
the chromosomes become more pronounced as rods or condensed
filamentous bodies (fig. 2) and the nucleolus disappears.

A subsequent breakdown of the nuclear envelope characterizes
the termination of the prophase stage (fig. 3). After the
nuclear envelope breaks down, the chromosomes are arranged
in the central region of the cell with no specific orienta—
tion; this marks the prometaphase stage. At this stage,
chromosome fibers are observed associated with the chromo—
some (figs. 4, 5 and 6). Since fibers were not observed in
the polar region of the cells at this stage, but were only

associated with or attached to chromosomes, it appears that

18

19

the chromosome fibers emanate from some region along the
chromosome. Light microscopic studies reveal that the
fibers are attached at the kinetochore region of the
chromosome. Observations of the living or fixed dividing
cell show that the kinetochore region precedes the other
regions of the chromosome in its movement toward the pole
(Bajer, 1951).

The migration of the chromosomes toward the center of
the cell and their alignment along the equatorial plane of
the cell marks the metaphase stage. At this stage, the
chromosome fibers are observed attached to the chromosome
and extending toward the polar region of the cell (figs. 7
and 8). In this investigation, the term chromosomal fibers
will be used to designate those fibers emanating from the
chromosome and extending toward the polar region. Those
fibers extending from one polar region to the other will be
termed the continuous fibers, and those fibers situated
between the separated chromatids, and which are attached to
them at either end, will be referred to as the interzonal
fibers.

What appears to be a single fiber when viewed with the
light microscope is, at the electron microscopic level,

actually a bundle of fibers, each with an average diameter

 

 

 

 

of 800 2.
each fibi
somal fit
700 X in
That t
and are a'
and 9. T?
in fig. 8
attachment
extend tow.-
Contin:
appear to c
of the chro
These Conti;
ChrOmOSOmal
In the C
afibrouS s:

 

fibers are r

Cells fixed

 

 

316a that S“!

9n”.
Heir low 91

 

20

of 800 A. Each fiber is made up of several fibrils with
each fibril measuring 125—150 A in diameter. The chromo-
somal fibrils observed by Sato (1958) ranged from 200 to
700 A in diameter.

{fixfizthe chromosomal fibers emanate from the chromosome
and are attached to the chromosome is suggested in figs. 8
and 9. The bundle of fibers emerging from the chromosomes
in fig. 8 appears to be very compact at the point of
attachment to the chromosome and less compact as they
extend toward the polar regions.

Continuous fibers are indicated in fig. 10, where they
appear to occupy a position tangential to certain portions
of the chromosomes observed in the section of this cell.
These continuous fibers are of the same dimensions as the
chromosomal fibers.

In the cells fixed with KMnO4 there is an indication of
a fibrous structure associated with the metaphase chromo-
some as suggested in figs. 11, 12 and 13. However, these
fibers are not as pronounced as those of the metaphase
cells fixed in the 0304 or the metallic salts described
earlier. The granules can be detected in the homogeneous

area that surrounds the less dense metaphase chromosomes.

Their low electron density is due to the fixation used.

21

In the cells fixed in 0504 and metallic salts, well-defined
chromosomal fibers were observed in this area. The endo-
plasmic reticulum and other cytoplasmic components are
better preserved with the KMnO4 fixation as also indicated
in figs. 11, 12 and 13. These cytoplasmic components
appear either to be migrating, or being pushed from the
metaphase plate toward the opposite ends of the cell for
future distribution in the daughter cells. That the loca-
tion of these components might be due to pushing is
suggested by their compactness around the periphery of the
homogeneous area of the metaphase plate. This compact
condition of the cytoplasmic components does not exist in
the interphase or prophase stages (figs. 14 and 15).

The separation of the sister chromatids characterizes
the beginning of anaphase (figs. 16 and 17). With this
separation the interzonal fibers are formed between them
and occupy the mid-region of the cell. With the progres-
sion of anaphase, the chromatids move toward the polar
regions, toward which the chromosome fibers are oriented
and in which they end (figs. l8, l9 and 20). At this stage
the dimensions of the chromosome fibers are the same as

those in the metaphase stage. The anaphase cell in fig. 19

was fixed in a formalin and metallic salt solution at a

 

 

pH 7..2. 5
to the cle
occupied t
pH does nc
dense line
are preser'
plasmic or;
shows the 1
interzonal
0fthe inte
“9 PrOpion
sions of t}:
0504 fixati'
in KMnO re'
4
Chromatids
fibers here
metallic sa

organelles

 

StitutiOn C
t€105)

haSe.

 

 

_—4-—‘ l

 

22

(pH 7.2. These anaphase chromosomes appear to be attached
to the clear lines, which probably represent the areas
occupied by the spindle fibers. The fixative used at this
.pH does not reveal the fibers as well defined, electron
dense lines. In figs. 21 and 22, the interzonal fibers
are preserved along with the chromosomes and other cyto-
plasmic organelles. This fixative (modified 0304) also
shows the fibrillar nature of the spindle (fig. 23). The
interzonal region is revealed with granules along the length
of the interzonal fibers after fixation in formalin, CrO3
and propionic acid as shown in fig. 24. The average dimen-
sions of the fibers here are the same as those fixed in the
0304 fixative. Close analysis of the anaphase cells fixed
in KMnO4 reveals interzonal fibers between the separated
chromatids as indicated in figs. 25 and 26. However, the
fibers here are not as pronounced as those revealed by the
metallic salts or the 0304 fixations. Here the cytoplasmic
organelles are packed in the opposite ends of the cell.

The grouping of the daughter chromosomes and the recon-
stitution of the nuclear envelope marks the beginning of

telophase. Along with nuclear reorganization, marked

changes occur in the central region of the cell. The mid—

region of the interzonal fibers becomes highly granular,

 

 

1.“. ‘3. a.’ r‘$.3r'e —!

and these

 

two defir
brane sys
which sta
the perip
sequent f1
interzonal
zonal fibe
a body tha
phragmopla:
m0plast 8p;
develOping

AS the

 

 

 

23

and these granules become aligned along the mid-region in
two definite rows (fig. 27), fusing to form a double mem—
brane system which marks the formation of the cell plate,
which starts in the center of the cell and continues toward
the periphery of the cell wall. The granulation and sub—
sequent fusion in the mid-region of the cell separates the
interzonal fibers into two units. At this stage the inter-
zonal fibers become detached from the chromatids and form

a body that is barrel-shaped in contour and is termed the
phragmoplast (fig. 28). The granular fibers of the phrag-
moplast appear to be oriented or migrating toward the
developing cell plate (figs. 29, 30 and 31).

As the granular fibers of the phragmoplast continue to
decrease in the mid—region of the interzonal area, the
double line of fused granules becomes more pronounced as
a double membranous system to form the cell plate of the
daughter cells (fig. 32). As cell plate development pro-
gresses, the space between the two membranes is increased
as shown in figs. 33 and 34. Each of the membranes meas-
ures about 400 A in diameter, and the less dense area
between them measures about 800-1000 A across its width.

This clear area between the two membranes contains dense

strands extending across its width; these probably mark

 

 

 

 

 

the inter<
cases (fig
continuous
pore or o;
plasmic co
In fig
and granule
These are 1
Porter and
The com
the regIO‘Jp
sion of the
Cell, with 1
interlonal
t

fie Separatj

31 +
- 3“» Cells

c~
“393- 9r».

 

3f
the Spin:

 

 

24

the intercellular connections, the plasmodesmata. In some
cases (fig. 33), the two membranes of the cell plate are
continuous with each other, along their length, forming a
pore or opening across its width, which provides a cyto—
plasmic connection between the two protoplasts.

In figs. 34 and 35, there are small spherical bodies
and granules associated with the developing cell plate.
These are reminiscent of the phragmosomes described by
Porter and Machado (1960).

The completion of the nuclear envelope formation around
the regrouped daughter chromosomes (fig. 36), and the exten-
sion of the membranous cell plate across the width of the
cell, with the disappearance of the granular fibrils in the
interzonal region, mark the end of the telophase stage, and
the separation of the daughter nuclei into two cells.

Many observations of colchicine—treated animal and
plant cells indicate that this chemical affects the spindle
fibers. Whether this is a partial or complete disruption
of the spindle depends on the concentration of the chemical
used.

In the examination of colchicine-treated seedlings, two
major types of configurations are observed in light micro-

sc0pic studies: (1) “scattered metaphase,” where the

 

chromo:
pro-me1
ball-sh
figurat
Bowen (L
that the
chemical
a less cc
concentra
fibers th
Obsers
cate that
the "clump;
tiOn of Co]

and wiISOn

 

 

 

25

chromosomes are spread about the cell, and (2) "clumped
pro-metaphase" where the chromosomes are arranged in a
ball-shape in the central region of the cell. These con—
figurations were previously described by Levan (1938),
Bowen (1953), and Hadder and Wilson (1957). It is assumed
that the "scatters" arise as a partial effect of the
chemical action on the spindle fibers and are produced by
a less concentrated dose of colchicine, while a greater
concentration completely destroys or disrupts the spindle
fibers thus producing a "pro—metephase clump."

Observations from light microscopic studies also indi-
cate that the "scatter" configurations appear first, with
the "clumps" appearing later, provided that the concentra-
tion of colchicine is high enough to produce them. Hadder
and Wilson (1957) maintained that the "scatters" do not
give rise to "clumps" because, at low concentrations, only
the "scatters" are noted and the "clumps" never appear,
regardless of the time of exposure of the seedlings to the
colchicine.

Electron microscopic observations of cells from seedlings
treated with 4.5 x 10-4 M of colchicine for 30 minutes also
reveal that "scatters" and ”clumps" occur (figs. 37 and 38).

The "clumped pro-metaphase" shows very little if any evidence

 

 

't‘ffi. “-r [7. w“

26

of spindle fibers while in the "scattered” condition definite
fibers are revealed attached to the chromosomes (fig. 37),
but not oriented as are the fibers in the control condition.
As the "scatters" and ”clumps" continue in the mitotic
process, the chromosomes become telemorphic (like the tele-
phase chromosomes in appearance and structure) and a nuclear
envelope is organized around them. At this stage the
restitution nucleus (fig. 39) contains four sets of chromo—
somes (28 chromosomes, the diploid being 14). As a result
of spindle disruption, there is no organization of the inter-
zonal fibers; therefore, no phragmoplast and cell plate
formation occurs, and cytokinesis does not take place. No

observable difference was detected in the cytoplasm of the

colchicine—treated cells from that of the control cells.

DISCUSSION

Until recently, the major studies of the spindle element
were made by means of the light and polarizing microscopes
and from these, conclusive evidence was obtained for the
reality of its fibrillar nature (Inoue, 1952). Thus far,
studies of the spindle, utilizing electron microscopic
techniques, have been relatively few, when one considers
the numerous studies on some of the other cellular com-
ponents such as mitochondria or plastids. The main reason
for the paucity of observation of the fine structure of the
spindle probably lies in the difficulty of cell preserva-
tion during the fixation and embedding process for electron
microscopic analysis. Although spindle preservation still
presents a problem, one can demonstrate spindle fibers by
using special fixation procedures as described earlier.

The micrographs described above present additional evidence
that the structural unit of the spindle fiber consists of
fibril units. The probability that the spindle elements
revealed here are complete artifacts seems remote, since
similar or identical spindle structures are observed in
various cells subjected to different fixation procedures.

In this investigation, observations reveal that the
spindle element consists of chromosomal fibers, interzonal

27

we. ‘r r—ny

 

 

fiber
T
somal,
origin
region
the StL
light rn
cells.
waS grea
Polar reg
birefringn
Vations if
Electron m
to be assoc
oward the J

that the Spi

 

28

fibers, a phragmoplast, and apparent continuous fibers.

The notion that the spindle fibers, i.e., the chromo-
somal, continuous and interzonal fibers, are of nuclear
origin and are derived specifically from the kinetochore
region of the chromosomes, has received added support from
the studies of Inoue (1953), who utilized the polarizing
light microscope to study spindle development in dividing
cells. The birefringence produced by the spindle fibers
was greatest near the chromosome and decreased toward the
polar regions. At metaphase and anaphase the fibers are
birefringent all the way to the poles. Since the obser-
vations in this study with the light microscope and the
electron microscope revealed that the spindle first appears
to be associated with the chromosomes and apparently grows
toward the polar region, and since there was no indication
that the spindle is formed in the polar region and extends
toward the chromosomes, the notion that the spindle fibers
originate from the kinetochore region of the chromosome
seems a reasonable one (Sato, 1958).

If the fibrous materials emanate from the entire area
of the kinetochore region, then as the chromosome is aligned
along the metaphase plate, the fibers perpendicular to the

equatorial plane would be oriented toward the polar region.

29

On the other hand, the fibers that extended into the hori-

zontal plane, i.e., parallel to the equatorial plane, would
have to undergo a reorientation in order to be directed
toward the polar region. If, however, the spindle element
is formed as a fluid or granular substance (fig. 4) and
this is oriented in a poleward direction during fibrillar
organization or polymerization, then this could probably
account for the continuous fibers that extend from one
polar region to the other which would then be tangential to
the lateral side of the chromosome.

That the spindle element could also emanate from regions
of the kinetochore that are perpendicular to the polar
regions should also be considered. In this condition,
whether the spindle is produced as ready—formed fibrils or
in a fluid or granular state, with fibrillar polymerization
occurring afterwards, there would still be polar orientation.

Whether the spindle element is produced as fibrils, as
a fluid, or in the form of granules with the subsequent
polymerization is not certain at this point. However, the
notion that the spindle fibers are produced in the form of
granules with a subsequent polymerization into fibrils seems
reasonable. This is indicated by the fact that, on close

examination of the point of fibril attachment to the

30

chromosome, granules appear along the length of the fibrils
that are embedded well into the chromosome area. Granules
are also arranged along the length of the chromosomal,
interzonal, and continuous fibers. Small, dense granular—
like fibrils were also described by Beams §t_§1, (1950),
Sedar and Wilson (1951), and more recently by Sato (1960),
as making up the spindle body.

The spindle fibers attached to the chromosomes at pro-
metaphase and persisting until late anaphase are referred
to as the chromosomal fibers. The combined light and
electron microscopic studies give evidence that these fibers
are attached to the chromosome at the kinetochore region and
extend to the polar regions (Sato, 1958). That the fibers
are directly attached to the chromosome is indicated in
fig. 7, and on close examination are seen embedded in the
chromosome (fig. 9).

With the poleward orientation of the chromosomal fibers,
the cytoplasmic organelles are concentrated in the opposite
ends of the cell and are pushed away from the area near the
nuclear boundary that they occupied at late prophase before
the nuclear envelope broke down. That the cytoplasmic com—
ponents are pushed by the spindle body is suggested by the

progression of the chromosomal fibers from the equatorial

 

‘_ p;‘na_

 

 

plate

ment t]
by some
ing of
cell co
cells.
It a
role in
Mota (19
from th
reaction
anaphase 1
ChrOmQSOnIE
fibers is
AlthOQ.
it is stilj
apparent eX
Gates a POs
ROWEVer,

SE

tores in t%

prodUCing s
are alSO as

attdChed Or

 

 

31

plate to the polar region. During this poleward develop—
ment the spindle body takes up areas that were once occupied
by some of the cytoplasmic components. This apparent push—
ing of the cytoplasmic organelles into opposite ends of the
cell could serve to distribute them within the daughter
cells.

It seems apparent that the chromosomal fibers play a
role in the movement of chromosomes to the polar regions.
Mota (1956) suggested that the release of spindle material
from the kinetochore possibly could cause a poleward
reaction which would push the chromosome and cause the
anaphase movement. Whether this poleward migration of the
chromosome is a push or pull action by the chromosomal
fibers is still uncertain.

Although the existence of continuous fibers is apparent,
it is still difficult to account for their formation. Their
apparent extension from one polar region to the other indi—
cates a possible formation at the polar region. This,
however, seems remote, since there are no observable struc—
tures in the polar region that give the appearance of
producing such fibers (Sato, 1958). Because these fibers
are also associated with the chromosomes and appear to be

attached or connected to them in a tangential fashion

32

(lateral side of the chromosome), it leaves one to speculate
that the spindle substance released from the lateral region
of the kinetochore might possibly account for the contin-
uous fibers. The substance would be released more or less
along a common region, and on polymerization would be oriented
toward both polar regions (see fig. 10), thus forming the
fibers that extend from one pole to the other.

On separation of the sister chromatids, fibers are
produced between them which occupy the mid-region and form
the interzonal fibers. Here, as in the formation of the
chromosomal fibers, there is a polymerization of fibrous
substance with a subsequent orientation parallel to the
longitudinal axis of the cell, making up the interzonal
fibers. These extend the length of the interzonal region
between the chromatids and are attached to them (fig. 24).
As anaphase progresses, the fibers are extended, increasing
the width and length of the interzonal region. As the
interzonal fibers increase in length, the chromosomal
fibers decrease in length, and the chromosomes are moved
toward the polar regions. Whether this increase in the
interzonal fibers is brought about at the expense of the

chromosomal fibers is not quite clear at this point. However,

33

the chromosomal fibers decrease and finally disappear as
the development of the interzonal fibers continues.

The separation of the interzonal fibrils into two units
by a horizontal orientation of granules across the mid-
region transforms this region into a barrel—shaped fibrillar
region, the phragmoplast, from which the cell plate will
develop (Sato, 1960). Along with these changes in the
mid-region of the cell, striking changes also take place
in the reforming nuclear regions, where the chromosomes
become regrouped and the nuclear envelope is organized
around them to form the daughter nuclei. As restitution
of the nuclei occurs, cytokinesis is also completed at
about the same time.

Wada (1955) maintained that the phragmoplast substance
is induced from the atractoplasm (spindle body) by hydra-
tion, and that during this change from atractoplasm into
the phragmoplast substance, the fibrils swell in this area.
The observations here revealed no appreciable difference in
the dimensions of fibrils of the phragmoplast from those of
the interzonal or chromosomal fibrils. Hewever, there
appears to be an increase in the number of granules in the
general interzonal area (fig. 28) and an ordered arrange-

ment of these granules along the mid-region of the phragmoplast.

 

barrE
para?
the re
T}?!

of the
Plate f‘
granules
formed b}
somal fib:
of the Chr
anaphase 5“
apparent in
the interzor
orientation
nid~region o

lateral dire
central forc
.ierizing gra‘

toward this

 

 

34

From these observations it seems probable that the
phragmoplast and the interzonal fibers are one and the
same, with the former resulting from changes in the fib-
rillar arrangement of the interzonal fibers to give a
barrel-shaped zone reduced in fibrillar structure. The
phragmoplast is located in the interzonal region between
the reforming daughter nuclei.

The occurrence of the granules across the mid-region
of the phragmoplast marks the early inception of cell
plate formation. In considering the origin of these
granules, one is tempted to speculate that they may be
formed by a depolymerization of the interzonal or chromo-
somal fibrils. This is inferred from the disappearance
of the chromosomal fibrils with the progression of late
anaphase and the inception of telophase. There is an
apparent increase in granularity throughout the length of
the interzonal fibers along with these processes. The
orientation of these granules into two lines along the
mid-region of the phragmoplast and their extension in a
lateral direction indicate the operation of some type of
central force, since the apparent diminishing or depoly-
merizing granular fibrils seem to be directed or oriented

toward this central region (see fig. 32).

35

As telophase progresses, the two lines of granules
along the mid-region of the phragmoplast fuse to form a
double membranous cell plate (see fig. 34). This condition
was also reported by Rozsa and Wyckoff (1950). These two
membranes appear to form simultaneously and appear as
double units from their inception. This development of
the cell plate starts at the central part of the phrag-
moplast and proceeds laterally toward the peripheral region
of the cell. As formation of the cell plate nears com—
pletion, the unit fibrils of the phragmoplast gradually
disappear. From the observations in this study it is
apparent that the cell plate, which is a double membranous
system separated by a clear area, is a product of: (1) a
granulation process of the interzonal fibrils, (2) a granu-
lar orientation at the mid—region, and (3) a granular fusion
to form a membranous system, the cell plate. On completion
of karyokinesis (nuclear division) and the separation of
the two protoplasts by cytokinesis, two daughter cells are
formed, completing active mitosis.

It is apparent from the observations of short-term
colchicine treatment of cells in mitosis, that the effect
is not an inhibition of mitosis, but rather an inhibition

of the spindle development and orientation or a disruption

a "—2.1. “a. 1'. rest: m ~ . .3... '~ W..- *

   

of th

with

repor

from

mater.

howeve

 

36

of the formed spindle fibrils. These observations agree
with those of Sedar and Wilson (1951) and also those
reported by Mazia (1956) on the isolated mitotic apparatus
from colchicine—treated eggs of the sea urchin. The
material that would normally form the spindle is present;
however, it does not acquire the typical fibrillar orien-
tation that is characteristic of the spindle element. It
seems probable that the protein molecules that build up
the spindle are altered in their composition or structure
by colchicine treatment. Mazia (1956) suggested that
protein molecules could still possibly form fibers through
oxidation of -SH- groups, but that these fibers would not
be capable of parallel orientation by hydrogen bonds.

It seems evident from the numerous studies of the
effect of colchicine on the dividing cell, that its
ability to alter the mitotic cycle lies in its direct
action on the spindle fibrils. That it affects the fibrils
directly is inferred from the observations of living
dividing cells treated with colchicine. When strong
concentrations (50 and 25 X 10-6 M) of colchicine were
added to mid and late anaphase, the spindle fibers were
destroyed, and the chromosomes stopped in their movement

to the poles, and the two groups of sister chromatids

» .3133-‘26'8113‘592

 

 

 

in

CO]

Spindle
develo‘n;
a greats
8‘"fiancee!
and the f
to the C375
flew

'elOP afz

 

 

37

intermingled, forming a single telophasic nucleus. When
concentrations of 2.5 x 10-6 M of colchicine were applied-
at the same stage, no detectable results were observed
(Eigsti and Dustin, 1955). Eigsti and Dustin also observed
that reduction of the metaphase spindle could be accom—
plished with lesser concentrations than those required for
anaphasic spindle reduction. From their observations of
dividing cells treated with colchicine, Gaulden and Carlson
(1951) concluded that the effectiveness of colchicine in
spindle destruction or interference with its further
development depends upon the concentration used, and that
a greater concentration is necessary to destroy the more
advanced spindle, i.e., at anaphase than at pro-metaphase;
and the form of a particular spindle is directly related
to the characteristic type of metaphasic pattern that will
develop after treatment, i.e., "scatters" or "clumps."
Observations of dividing cells treated with varying
concentrations of colchicine indicate that low doses cause
a "scattering" effect, while the high doses produce complete
spindle destruction, and therefore "clumps" are produced
(Eigstxi and Dustin, 1955; Hadder and Wilson, 1957). In
the "clumps" observed in this study, there were no well-
defined spindle fibers detected, while in the case of

"scatters," definite fibrils were observed.

tion
chrom‘
p019we
elemer‘
fibers
resu1t
continu
typical
become t
an inter.
a tetrapl

are disru‘

h‘r‘

airomosome
separation
complements
on

e cell in

Spindle fibr

 

38

The destruction of spindle fibers prevents the forma—
tion of the metaphase plate and the subsequent movement of
chromosomes to the polar regions. This failure of the
poleward movement in the absence of an oriented spindle
element indicates the important role played by the spindle
fibers in the movement of chromosomes to the poles. As a
result of this fibrillar disorganization, the chromosomes
continue their morphological cycle in_§itg, Although the
typical telophase stage is not formed, the chromosomes
become telomorphic (become like telophase chromosomes) and
an interphase nucleus is eventually reconstituted which is
a tetraploid. While the molecules of the spindle fibrils
are disrupted and the fibrillar orientation affected,
chromosome replication is not affected, and the chromatid
separation is not impaired. Therefore, the chromosome
complements for the two daughter cells are present in the
one cell in which cytokinesis failed to occur because of

spindle fibrillar destruction by colchicine treatment.

\

. “A."
... 3:."
. .5 ’1'; VH‘!h———

 

357
Ad!

'u

0‘
O
y...

consi51
('3) con
rise to
are com"
diameter.
Which in
3. T}
0f the C1517
intergonal
4. The
53059 the m

:use to fOrm

  
 

‘V,
h"

0 protopla-

SUMMARY

1. The fine structure of the spindle element is revealed

K Cr 0 and

by the use of certain metallic salts as CdClz, 2 2 7,

PbClz. The spindle fibers were also demonstrated by using

0304 at various pH ranges while still preserving other
cellular organelles.

2. In gisum_meristematic cells the spindle element
consists of (l) chromosomal fibers, (2) interzonal fibers,
(3) continuous fibers, and (4) a phragmoplast which gives
rise to the cell plate. The fibers measure about 800 X and
are composed of unit fibrils which measure 125 — 150 S in
diameter. The fibers are grouped together to form bundles
which in the light microscope appear as a single unit.

3. The fibers originate from the kinetochore region
of the chromosome. The phragmoplast develops from the
interzonal fibers and gives rise to the new cell plate.

4. The cell plate is formed as two lines of granules
along the mid—region of the phragmoplast. These granules
fuse to form a double membranous system which separates the
two protoplasts into the two daughter cells at cytokinesis.

5. On treating the dividing cell with a threshold

concentration of colchicine the fibrillar orientation of

the fibers is disrupted, thereby destroying the spindle

39

‘ .03..)- r..

 

 

 

H
N.

not
orie
colc.

:v' t 0k

 

4O

fibers as an organized system. Because of spindle disrup-
tion the developing chromosomal and interzonal fibers are
not formed, neither do the formed fibers maintain their
orientation when treated with such concentrations of
colchicine. Therefore, the cell plate does not develop and

cytokinesis fails to occur.

 

 

" " "e ‘t— '- I
.r.?"«"n-_—u ,

 

Andre

Bajer,

me.

 

BIBLIOGRAPHY

Amano, S. 1957. The structure of the centriole and spindle
body as observed under the electron and phase micro—
scopes. Cytologia, 22:2—193.

Andrews, F. M. 1915. Die wirkung der Zentrifugalkraft auf
Pflanzen. Fahrt. Wiss. Bot., 56:221—253.

Bajer, A. 1951. Studies on spindle and chromosome move—
ment. Acta. Soc. Bot. Pol., 21:95—111.

1957. Cine-micrographic studies on mitosis in endo—
sperm. III. The origin of the mitotic spindle. Exp.
Cell Res., 13:493—502.

Beams, H. W. and R. L. King. 1936. The effect of ultra-
centrifugation upon chick embryonic cells, with special
reference to the resting nucleus and the mitotic
spindle. Biol. Bull., 71:188—198.

Beams, H. W., T. C. Evans, V. V. Breeman and W. W. Baker.
1950. Electron microscopic studies on structure of
the mitotic figure. Proc. Soc. Exp. Biol. and Med.,
74:717-720.

Becker, W. A. 1938. Recent investigations in_yiyg on the
division of the plant cell. Bot. Rev., 4:446-472.

Belar, K. 1929a. Beitrage zur Kausalanalyse der Mitose.
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~. w”: m

 

ce:

mat
Bessis,
Cent
étud
13:3'
Meier, i

Zellf

 

 

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V 7 I" ' IL’.‘ A: u H

 

EXPLANATION OF FIGURES

KEY TO ABBREVIATIONS

Extra Cellular Components

- cell wall

W

pd - plasmodesmata

pm — plasma membrane

Cytoplasmic Components

er

PP

pr

endoplasmic reticulum
dictyosome

mitochondria

proplastid

cytoplasmic inclusion body
vacuole

tonoplast

polar region

Nuclear Components

 

ne - nuclear envelope
n - nucleus
ch — chromatin material
nu - nucleolus

Spindle Element
chf — chromosomal fiber
if - interzonal fiber
of - continuous fiber
cp - cell plate

51

52

Fig. 1. A low—power image of a longitudinal section through
the meristematic region of a root tip, showing the inter-
phase and prophase stages. The clear areas between the
cells represent the cell wall (w), while the dense line
that forms a boundary around the cytoplasm is the plasma
membrane (pm). The mitochondria and proplastids are shown
as oval or elongated dense bodies (see arrows). Because

of low magnification it is difficult to differentiate
between these two organelles. The endoplasmic reticulum
(er) is shown as a dense membranous system distributed
throughout the cytoplasm. Small dense particles are also
distributed through the cytoplasm forming a granular ground
substance. The nuclear envelope (ne) is shown as a dense
double membrane, the chromatin material (ch) in the nucleus
(n) is shown as dense bodies and the nucleolus (nu) as a
dense oval body. Fixation in 1% 050 . Approximately X

4
8,000.

‘-
. g, A.

1"
V r
('A

0’:
_ or
i .
'-
’t

r:
.

 

 

——A-:-IIIII

54

Fig. 2. A late prophase with some of the chromosomes (ch)
located at the periphery of the nucleus and adjacent to

the nuclear envelope (ne). Fixation in 1% 0504. Approxi—

mately X 20,800.

55

 

Figure 2

 

y. ‘¥.WZ‘—9 - v

56

Fig. 3. Longitudinal section through interphase and late
prophase cells. The nucleolus (nu) of the interphase cell
is shown as a spherical dense body with a less dense cen—
tral area (x). Chromatin (ch) material appears in both
cells as dense bodies. The nuclear envelope (ne) of the
late prophase cell appears to be in the process of breaking
down (see arrows). Fixation in 10% formalin and post fixed

in 1% 0504. Approximately X 10,800.

I» - v 5 .
o ' > .‘
$ ‘ \“
05's E Q
\ ‘ 1‘ ' A

“1:
U" .
4 .- ’ S‘s-fl

 

Figure 3

 

 

~ '7r.1.»g

 

58

Fig. 4. A diagram depicting a probable method of spindle
fiber production by the kinetochore region of the chromo-
some. A, the spindle substance is produced along the kine-
tochore region as granules, B, polymerization of granules
into fibrils, C, orientation of fibrils toward polar regions,
to give chromosomal fibers (chf), D, X.S. of kinetochore
region depicting polymerization and orientation of granules
into fibrils, with the fibrils at Cf probably giving rise —
to the continuous fibers, E, formation of interzonal fibers
(if) at the separation of the sister chromatids during
early anaphase, F, late anaphase, where the chromatids have
moved closer to the polar regions, G, early telophase with
phragmoplast (see arrows), and two rows of granules across
the mid-region marking the inception of cell plate develop-
ment (cp), H, late telophase with diminishing phragmoplast
and membranous cell plate, and I, completion of karyokinesis

and cytokinesis with the production of two daughter cells.

59

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60

Fig. 5. (A) A light micrograph showing a metaphase with
chromosomal fibers (chf) extending from the chromosome
(ch) to the poles. (B) shows a telophase with the inter-
zonal fibers (if) and a clear line across the mid—region
indicating the formation of the cell plate (see arrows).

Fixation in Bouin's fixative. X 3,500.

 

 

62

Fig. 6. Section showing chromosomal fibers (chf) emanating
from pro-metaphse chromosomes (ch), and cell wall (w).

Fixation in 2% K2Cr207. Approximately X 13,200.

 

 

 

 

64

Fig. 7. Portion of meristem cell showing metaphase plate
with chromosomal fibers (chf) attached to chromosomes (oh),
and extending to p01ar regions (pr), plasma membrane (pm)
and cell wall (w). Fixation 1% PbCl2 and 10% formalin.

Approximately X 13,200.

 

Figure 7

 

 

 

66

Fig. 8. Section through metaphase plate showing bundle of

chromosomal fibers (chf), see arrows. Fixation in 2% Cr03.

Approximately X 13,200.

 

 

 

68

Fig. 9. Portion of fig. 7 enlarged to show attachment of

fibers (chf) to chromosome

of the fibers (see arrows).

(ch) and the granular appearance

Approximately X 84,000.

 

Figure 9

 

 

 

 

 

70

Fig. 10. Portion of a metaphase plate showing what appear
to be continuous fibers (cf) along with chromosomal fibers

(chf). Fixation in 2% CdClz. Approximately X 17,600.

 

Figure IO

 

 

72

Fig. 11. A tangential section across metaphase plate of
meristem cell, in which the chromosomes (ch) appear as
low—electron dense areas across the cell, granules are
shown in the spindle area (see arrows), mitochondria (m),
endoplasmic reticulum (er), cytoplasmic inclusion bodies
(i), proplastids (pp), plasma membrane (pm), plasmodes-
mata (pd), cell wall (w). Fixation in 2% KMnO . Approxi—

4
mately X 13,200.

’71.! k 3‘

 

Figure ll

 

_ u—mmmf ‘.:§,~“g.;.. ,3. .. _ __

 

 

74

Fig. 12. This section shows a metaphase plate with bits
of broken-down nuclear envelope (ne) and endoplasmic
reticulum (er) surrounding the chromosomes (ch) that are
in an area containing granules (see arrows). Dictyosomes
(d), mitochondria (m), and other cytoplasmic inclusions

are also shown. 2% KMnO4 fixation. Approximately X 13,200.

 

76

Fig. 13. Section through the central part of a meristem
cell that shows chromosomes (ch), aligned along the equa-
torial plane that are surrounded by spindle area (see
arrows), bits of nuclear envelope (ne), endoplasmic reticu-
lum (er), proplastids (pp), cytoplasmic inclusions (i),
dictyosomes (d), and other unidentified particles are also
shown, along with the plasma membrane (pm), plasmodesmata
(pd) and cell wall (w). Fixation is in a 2% KMnO solution.

4
Approximately X 10,800.

77

 

Figure l3

 

 

 

 

 

 

78

Fig. 14. Longitudinal section of interphase cell which
shows the distribution of cytoplasmic components. Note
the loose distribution of components here, in contrast to
the compactness of components in the opposite ends of the
cell as in the case of the metaphase stage. The plasmo-
desmata (pd), plasma membrane (pm), along with the dictyo—
somes (d), proplastids (pp), mitochondria (m), and endo-
plasmic reticulum (er) are shown. The nuclear envelope
(ne) surrounds the homogeneous nucleus (n) which has a
wavy appearance due to knife marks from sectioning. The
endoplasmic reticulum (er) appears to be connected to the
nuclear envelope (see arrow). Fixation in a 2% KMnO and

4
a 2% PTA solution. Approximately X 13,200.

 

 

 

 

 

 

80

Fig. 15. This micrograph shows a longitudinal section of
an interphase cell that reveals the distribution of cyto-

plasmic components after 080 fixation in contrast to fig.

4
13, which is fixed in KMnO4. Note that the distribution
of components is similar to that of fig. 13; however, the
nuclear components appear as dense granular or rod-like
bodies. Vesicles are also distributed throughout the

cytoplasm (arrows). The cellular components are marked

as before. Approximately X 13,200.

 

 

Figure IS

 

 

 

 

82

 

Fig. 16. This section shows the association of chromo-
somal fibers (chf) and the interzonal fibers (if) with

the separating chromatids (ch). Vacuoles (v) with a dense
membrane surrounding them, the tonoplast (t). Dense
inclusion bodies (i) and the plasma membrane (pm) are well
preserved. Fixation in modified OsO . Approximately

4
X 5,600.

 

Figure 16

 

 

 

84

Fig. 17. A portion of a meristematic cell which shows a
section through an early anaphase with chromosomal fibers
(chf), interzonal fibers (if), and dense chromosomes (ch).

Fixation in 1% 0504. Approximately X 20,800.

85

 

 

Figure I?

 

86

Fig. 18. This section shows the relation of chromosome
(ch) to spindle (see arrows) and to the polar regions (pr),
toward which the chromosomal fibers (chf) are oriented

during anaphase. Fixation 2% Cr03. Approximately X 13,200.

 

Ihd

 

88

Fig. 19. Micrograph showing a section through the central
part of a meristematic cell in which the anaphase chromo-
somes are associated with clear lines. These probably
represent the areas occupied by the spindle fibers, but
with the fixative used, the fibers are not revealed as well
defined electron dense lines. These clear lines (see
arrows) occupy areas similar to the fibers demonstrated in
fig. 18. The clear lines extend to the polar regions which
contain a dense body (X). Whether these dense bodies at
the polar regions are portions of chromosomes or inclusion
bodies is not clear at this point. Vacuoles (v) occur in
the cytoplasmic area surrounding the spindle body. Fixation
in 2% K Cr 0 and 10% formalin at pH 7.2. Approximately

2 2 7
X 13,200.

89

   

Figure 19

h.

 

90

Fig. 20. This micrograph shows a section through a cell
at mid-anaphase in which the chromosomal fibers (chf) are
oriented toward and ending in the polar region (pr). The
chromosomes (ch) are shown with lagging arms (X) and with
kinetochores directed toward the poles. The interzonal
fibers (if) are located between the separated chromatids.

Fixation in 2% CrO3. Approximately X 13,200.

 

9i

 

 

 

 

 

‘A‘!’ ”'5'”: firm‘wzs‘ ”éfltmfi‘q‘fl" --e‘:'."-' “5 l v" '

92

Fig. 21. Portion of an anaphase cell showing cytoplasmic
organelles being pushed to both ends of the cell (see
arrows), chromosomes (ch), interzonal fibers (if), pro—
plastids (pp), and mitochondria (m). Dense granules appear
throughout the cytoplasm. Fixation in modified 0504 at

pH 5.6. Approximately X 13,200.

 

Figure

 

 

 

94

Fig. 22. This micrograph shows a longitudinal section
through a late anaphase stage, from the meristematic
region of the root tip. The vacuoles (v) contain dense
granular inclusion bodies (i). The chromosomes (ch)
appear as dense bodies with the chromosomal fibers (chf)
and interzonal fibers (if) attached to them (see arrows).

Fixation in 1% 0304 and 1% CdC12. Approximately X 20,800.

95

 

" . t ‘ 5‘ ‘
I . .i" ‘ fi -.
‘k‘ ' a, ‘ .
' - I ' I I}: Kl.» ‘
. ~ ’ ..J‘: ‘ ‘ J; a I . ‘ .'

 

Figure 22

 

96

Fig. 23. An enlarged view of fig. 21 which shows the
fibrillar nature of the interzonal fibers (if). A bundle
consists of several fibers (see overlay), while the
fibrils make up the fibers (overlay). Fixation in 050 .

4
Approximately X 63,600.

'r.‘

 

 

 

 

 

 

h”

 

' 1 ~13, 42.7“. v r ’."l|d.1"fs.

Figure 23

98

Fig. 24. A section through an anaphase cell which shows
chromosomes (ch) with lagging arms (see arrows), and
granules along the length of the interzonal fibers (if).
Fixation in K2Cr O and 10% formalin. Approximately

2 7
X 25,600.

 

 

Figure 24

_.....aw""~‘< 15‘": 5* ..«.rq_7‘.

 

100

Fig. 25. This micrograph shows an anaphase cell after

fixation in KMn04. The chromosomes (ch) appear as non-

electron dense areas. The interzonal region (area between
the separated chromatids) contains granules (see arrows).

This fixative (a 2% KMnO4) does not preserve the spindle

fibers as very dense lines as does the 0504 and the CrO3.

Note the compactness of the cytoplasmic components in the

opposite ends of the cell (X1 and X2). Approximately

X 10,800.

 

 

 

~ rar- wr‘. “=‘ficp M ""2- -wuu—.-— .. I_"~~'~""~.W‘W§I"' ' ' "

 

 

102

Fig. 26. Section through an anaphase cell that shows bits
of either the broken—down nuclear envelope (ne) or the
endoplasmic reticulum (er) around the area surrounding the
anaphase chromosomes (ch), the dense streak across the cell
is a fold in the plastic film on which the section is

being supported. Note granules in the interzonal region

(arrows). Fixation as in fig. 25. Approximately X 10,800.

 

 

~.- 't WW -~, w--— . . its: ~.._'

104

Fig. 27. A portion of an early telophase with two rows
of granules across the mid—region (see arrows) of the cell.
This marks the inception of cell plate formation. Chromo—
somes (ch) are in the process of regrouping for daughter
nuclear formation. Fixation is in a modified OsO .

4
Approximately X 20,800.

“.

I a

$.l‘

S

.
,v.
. m“
I
o

~

. .
l

I

 

Figure 27

a,“

 

 

 

 

106

Fig. 28. This section through the cortical region of the
meristem shows a telophase cell with regrouping of chroma-
tids (ch) to form the daughter nuclei (the dense particle
near the left side of the lower regrouping nucleus is a
piece of debris on the section). The mid-region contains
a "barrel—shaped” phragmoplast (see arrows) with two
apparent rows of granules across its mid—region marking
the cell plate formation (cp). The mitochondria (m)
proplastids (pp), vacuoles (v), plasma membrane (pm) and
cell wall (w) are also preserved. Fixation in 1% 050 .

4
Approximately X 13,200.

 

 

Figure 28

 

 

 

' " ‘WM‘"*~~"_— l

 

108

Fig. 29. Section showing barrel-shaped phragmoplast (see
arrows) regrouped chromosomes (ch) developing cell plate
(cp). Note the adjacent cell to left where the cell plate
(cp) has extended to the cell wall (w). Fixation in 2%

CdC12. Approximately X 8,000.

 

Figure 29

110

Fig. 30. A longitudinal section through a telophase which
shows the diminishing fibrils of the phragmoplast (see
arrows), and the cell plate (cp). Fixation in 10% formalin

and 1% 0304. Approximately X 8,000.

 

O
3
e
r
U
9
I.
L-

 

112

Fig. 31. Portion of telophase cell where the fibrils of

the phragmoplast are increasing in granularity

(see arrows).

Fixation in 2% K2Cr207 and 10% formalin. Approximately X

10,800.

 

 

Figure 3]

i.. -

l

 

“:5!" A ‘

 

 

 

114

Fig. 32. This micrograph shows a section through a telo-
phase cell where the cell plate (cp) extends the entire
width of the cell (see arrows) separating the daughter
nuclei (n). Fixation in 2% K2Cr207 and 10% formalin.
Approximately X 13,200.

 

 

 

116

Fig. 33. Portion of late telophase to show diminishing
fibers of phragmoplast (see arrows) and extension of cell
plate (cp) across width of the cell. At X the two mem-
branes of the developing cell plate are continuous with
each other forming an opening across its width, which
provides a cytoplasmic connection between the two proto—

plasts. Fixation as in fig. 32. Approximately X 20,800.

 

Figure 33

 

 

.-w—vm— v 3,,— ‘- ~ '. w- ~ :‘w nWW‘:¢Wr~r in v -.-i mm~wen vs“ "1‘ -' flaws-F" 'V"’ ' "em-r ' -"r..-‘ ‘ ‘ fimszfl
.

118

Fig. 34. Longitudinal section through the peripheral
region of a telophase cell (missing daughter nuclei
altogether), cell plate (cp), plasmodesmata (pd),
inclusion bodies (i) and other cytoplasmic components
are shown as described before. Fixation in 2% KMnO .

4
Approximately X 13,200.

 

 

Figure 3h

 

 

120

Fig. 35. Section through one end of a telophase cell which
shows the reorganization of the nuclear envelope (ne),
inclusion bodies (i), mitochondria (m), proplastids (pp)
and small dense granules (see arrows) associated with the
developing cell plate (cp). Fixation in 2% KMnO4.
Approximately X 13,200.

 

 

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122

Fig. 36. Longitudinal section through telophase cell
showing regrouped chromosomes (ch) within the newly re-
organizing daughter nuclei (n). NCte nuclear envelope
(ne) reformation, cell plate (ce) in center of cell with
mitochondria (m), proplastids (pp), endoplasmic reticulum
(er) and other cytoplasmic organelles appearing in the
central region (see arrows) of the cell. KMnO fixation.

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Approximately X 10,800.

 

 

 

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124

Fig. 37. Portion of dividing cell showing "scattered"
arrangement of chromosomes (ch) with bits of attached
chromosomal fibers (chf, see arrows) after treatment with
a 4.5 X 10—4 M of colchicine for 30 minutes and recovered

for 6 hours. Fixation in 0804. Approximately X 20,800.

 

Figure 37

 

 

 

126

Fig. 38. This micrograph shows a section through a pro-
metaphase "clump." As a result of treatment-with 4.5 X
10-4 M of colchicine for 30 minutes and a recovery of 6
hours the usual metaphase plate is not established (as in
figs. 4, 6 and 12), because the chromosomal fibers are
destroyed, preventing this arrangement from taking place.
Here the chromosomes (ch) appear as dense bodies.

Fixation in 2% CdCl2 and 1% 0504. Approximately X 20,800.

 

 

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128

Fig. 39. Section through a tetraploid interphase cell.

As a result of fibrillar disorganization by colchicine
treatment, the chromosomes continue their morphological
cycle ig_§i;uj becoming telomorphic and the interphase
nucleus is eventually reconstituted which is a tetraploid.
Here the chromatin (ch) and nucleoli (nu) appear as dense
bodies within the nuclear area (n). Fixation in 2% CrO

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and formalin at pH 7.2. Approximately X 13,200.

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