...1;
V
.
3
67
ORNL
UNCLASSIFIED
LE
'




ORNn-P-67 ...
Submitted to: p.
Experimental Cell Research
JUL 1 0 1964
MASTER
.
Mitochondrial Division in Phytohaemagglutinin-Stimulated Human Lymphocytes
Hiroshi Yamamoto
Biology Division, Oak Ridge National laboratory, Oak Ridge, Tennessee

This paper was submitted for publication
in the open literaturg at loan' months
prior to the issuance date ni thio Micro-
card. Sinc? the U.S.A.E.C. has no evi-
dence that it has boen published, the pa-
per is being distributed in Microcard
form as a preprint.
-LEGAL NOTICE
The report no prepared un account of Goverment sponsored work. Notchor the United
Histo, por the counselor, mor me porno actions on behall of the Coualeatoa:
A. Kuo wymruty or reprenatation, emprend or lapte, nu repect to accy-
recy, completenes, or we human of the talorutou could uwi report, or that the we
of uy taformation, appunto, awd, or proceed towadu o report wy hot latta
printoly owned rhonco; or
D. And, may lasilium mu roopust to the use of, or for damage rending trou the
w of uw laborwalon, appunto, method, or proces declared laws report.
As ww u the bore, "roro Ktha hell al Comnim " faciun
Noys or contractor of the Con ton, or aplogue of much contractor, to the em ut
much emplogne or contructor al the Counlodlou, or planned much contractor porno,
domuinto, or prondes acc. to. May leformation pumant to do inployum or contract
vith the Conwealoo, or Ms explogwat vu such contractor.

Running head: Mitochondrial Division in Phytohaemagglutinin-Stimulated
Human Lymphocytes
Send proof to: Dr. Hiroshi Yamamoto
Biology Division
Oak Ridge National Laboratory
P. 0. Box Y.
Oak Ridge, Tennessee 37831
j.
.
-
--
MI TOCHONDRIAL DIVISION IN PHYTOHAEMAGGLUTININ-STIMULATED HUMAN LYMPHOCYTES
HIROSHI YAMAMOTO
Biology Division, Oak Ridge National Laboratory*
Oak Ridge, Tennessee
Introduction:
The mitochondrion consists of an outer membrane, outer
chamber, inner membrane, cristae mi tochondriales, inner chamber with
matrix, and dense granules (1). Recent electronmicroscopic studies
using negative staining show that the structura, elements of the
mi tochondria are composeči of elementary jarticles with a cylindrical
stalk, and mesolayer, which is made up of lipid-protein netwerk (2, 3).
Each elementary particle contains the complete electron-transfer chain
for the oxidation of succinate or DPNH by molecular oxygen (4). There
have been many speculations about mitochondrial origin. Fundamentally,
they are classified into two groups: one which includes those formed
mitochondrial precursor structures (microbodies, nuclear envelope,
endoplasmic reticulum, and plasma membrane); the other includes those
formed by pre-existing mitochondria.
Autoradiographic data (5), using Hº- or c+*-choline, has shown that
the most likely interpretation of studies with Neurospora that the
mitochondrial mass is increased by building on the existing structural
framework, and that the number of individuals in the population is
increased by division of mitochondria. Other data (6) on the weight
distribution of isolated subcellular particles from normal rat liver, and
mer...
on classification by morphological criteria shows that mitochondrial fraction
is divided into two groups, also suggesting that mitochondria di.vide.
The experiments reported here attempt to interprete the morphological ·
alteration during mitochondrial division in phytonaemagglutinin-stimulated
.
17
*Research sponsored by the U.S. Atomic Energy Commission under contract with
the Union Carbide Corporatia.
human lymphocyte cultures, and to relate these alteration to the mechanism
by which new mitochondria are formed .
Materials and Methods: Adult human peripheral leukocytes were cultured
with Mixture 199 supplemented with human serum and phytohaemagglutinin.
Every six hours after initiation of the cultures, the cells from one of
a group of repcate cultures were fixed with 4 per cent cold potassium permana:
ganate for 1.5 hour, embbeded in epoxy resin, sectioned on a Porter-Bloom
microtome, and examined with an RCA EMUŽE electron microscope .
The measurement of sizes in major and minor axis of mitochondria
on the electronmicrographs, which were picked out at random, were done
with an automatic priuting measuring device, consisting of a linear
resistor connected to a pair of dividers in such a way as to provide
2 voltage proportional to the separation of the divider prints. This
voltage is read out by a digital voltmeter and automatically printed
on tapa.
Results: Plastic transformation of ly.phocytes. - In differential cell
counts (7), the number of polymorphonuclear granulocytes and small
lymphocytes decreased during the first 36 hours of culture life. No
mitotic cells were seen until 48 hours after initiation of the cultures,
which agrees with the autoradiographic data of Bender and Prescott (8).
Using acridine orange staining and Kariometry (7), it was observed that
nuclear and cellular size and average nucleolear number of the lymphocytes
increased during the course of the culture. The large mononuclear cells
showed prominent nucleoli, most of which had clear nucleolonema .
HEIM
..
Prominent granular reddish-orange cytoplasmic fluorescence was seen
in the large mononuclear cells, which are the cells which subsequently
undergo mitosis in these cultures, and 18 suggestive of extensive
protein synthesizing capabilities.
Silze measurements of mitochondria. - Most of the mitochondria 10
cells of the lymphocyte series are oval in the sections.
The major
measured with the recording dividers .
A plot of major axis length against minor axds length (Fig. 1)
shows that there are two size classes of mitochondria. Fig. 2 and 3
show the histograins of frequency distribution of mitochondrial major-
and minor axis.
In the intermediate type cells, no remarkable elongation of the
major axis of the mitochondria is seen. On the other hand, . some of
the mitochondria in the interphase of large mononuclear cells and in
mitotic cells show an increase in length, especially in major axis.
The mean sizes, with standard errors, of the mitochondria in the human
lymphocyte cultures are shown in Table 1. The results suggest that the
enlargement, especially elongation, of mitochondria is induced in the
mitotic phase, and perhaps even in the interphase, of the proliferating
cells.
Structural alteration of mitochondria. - A portion of the cytoplasm in
the interphase of a large mononuclear cell derived from a lymphocyte, with
markedly elongated mitochondria and a few oval shaped small mitochondria,
is shown at the right side of the electronmi crograph (Fig. 4). We recognize
1
Vi!
.
small bubbles in the outer chamber, 1.e. between the outer- and inner-membrane.
These bubbles are often observed at the junct..on of cristae mitochondiales
(indicated by arrows). This phenomenon suggests the replication of
mi tochondrial cristae prior to mitochondrial division in cells which
undergo cellular division. Fig. 5 shows the diagrammatic representation
of the scheme of mitochondrial division in phytohaemagglutinin-stimulated
human lymphocytes suggested by the electronmicrographs.
Discussion: The enlargement or swelling of mitochondria has been shown to
be an early sign of cellular damage in various physiological and pathological
condition (9, 10, 11). In tissue cultures supplemented with acid medium
(10), or with detergent (11), the swelling of mitochondria has also been
reported. In the normal dividing cells, multiplication of mitochondria
is an obvious necessity. The data, derived by electronmicroscopy and by
isolation techniques (6), have shown that mitochondria in the normal rat
liver increase their weight by linear growth, and then divide, and also
that the division is not necessarily symmetrical. Radioautographic
analyses (5) have indicated mitochondrial division. My present data
also suggest the same phenomenon, and mitochondrial divisions are seen
both in the interphase and mitotic phase of the proliferating cells in
our leukocyte cultures. Moreover, the ultrafine structure of mitochcndria
with negative staining has been clarified and a macromolecular model of
mitochondrial structure has been derived (2, 3, 12). With these data and
my electron microscopic data of dividing mitochondria,'the macromolecular
model of the replication of mitochondrial cristae in the mitochondrial
division may be drawn as in Fig. 6.
REFERINCE:3
1. Palade, M. G., J. Biophys. Biochem. Cytol. l, 567, (1955).
2. Fernandez-Moran, H., The Structure of the Eye, Academic Press,
New York, p. 521, (1961).
3. Green, D. E., and Fleischer, S., Biochim. Biophys. Acta, 70, 554, (1960).
4. Blair, P., Oda, T., Green, D. E., and Fernandez-Moran, H., Biochemistry,
2, 756, (1963).
5. Luck, D. J. L., J. Cell Biol. 16, 483, (1963).
6. Bahr, G. F., and Zeitler, E., J. Cell Biol. 15, 489, (1962).
Yamamoto;.9., unpublished.
8. Bender, M. A, and Prescott, D. M., Exptl. Cell Research 29, 430, (1963).
9. Opie, E. L., J. Exptl. Med. 87, 425, (1948).
10. Cowory, E. V., Arch. Pathol. Lab. Med. 1, 237, (1926).
11. Frédéric, J., Ann. N. Y. Acad. Sci. 58, 1246, (1954).
12. Parsons, D. F., Science 140, 985, (1963).
.
..
.
1
org
Table 1
RELATIVE SIZES OF MITOCHONDRIA IN PHYTOHA EMAGGLUTININ-STIMULATED
HUMAN LYMPHOCYTES IN MICRA
MEAN SIZE AND STANDARD ERROR
CELL-TYPE
PHASE
NO. OF MITO.
MEASURED
.
MAJOR AXTS.
MINOR AXIS
0.56 + 0.07
0.33 + 0.03
INTERMEDIATE TYPE
INTERPHASE
.
15
INTERPHASE
.
.
. 73
0.85 +0.05
0.46 + 0.02
BLAST-LIKE CELLS ...
1.24 + 0.09
0.72 + 0.04
MITOTIC PHASE
## standard dovtetterna
...
Fig. 1
PLOT OF MAJOR AXIS AGAINST MINOR AXIS OF MITOCHONDRIA IN PHYTOHADU GOLOTININ.
STIMULATED HUMAN LOPHOCYTES
2.0
:
.
.
.
.
.
MAJOR AXIS OF MITOCHONDRIA MEASURED
.
..
.
0.5
1.0 (u)
-
-
MINOR AXIS OF MITOCHONDRIA MEASURED
Fig. 2
HISTOGRAM OF SIZE DISTRIBUTION OF MITOCHONDRIAL MINOR AXIS IN
PHYTOHA EMU GOLOTININ-STDAULATED HUMAN LOPHOCYTES
•

i.
.
TIL
in intornodiato colls
in interphase of blast-11ko colls
in dtotio phase of blast-liko oolls
total
Sobot X
h
20
20. F.
.
UF IV
Temiri
H
FREQUENCY
TERKILVIH. 71-
sistemom 10:57
RegVi50.tary
i
325H25Vasa
7870
0.5 :.
MINOR AXIS OF MITOCHONDRIA MEASURED
Dj
HISTGRAM OF SIZE DISTRIBUTION OF MITOCHONDRIAL MAJOR AXIS IN PHYTOHA EMAGGLUTININ-STIMULAT E
HUMAN LYMPHOCYTES
.'
.

XKSDIERS
2
FREQUENCY
pressuroinut saw
22.5
TSIUMKLORISTMAS
Pique 30X22
WRTHER
So
P2412422.
!
*
0.5
1.5
2.0
(u)
MAJOR AXIS OF MITOCHONDRIA MEASURED
.
Fig. 4. Electron ad crograph of the markedly elongated mitochondria
in phytobaemagglutinin-stimulated human lymphocyte. In the outer
chamber, various sized bubbles (indicated by arrows) are depicted.
X 84,000
-
-
-
..
.
.
.
i 20
.
.
به
-
..
.
.
. .
..
"
.
# تنجح

AC
tary .
.
끼
​AIA
.
.
.
5..
RX :
Fig. 5
SCHEMATIC DRAWING OF THE MITOCHONDRIAL DIVISION
IN THE PHYTOHUADAGOLOTININ STIMOLAT ED HUMAN LOPHOCYTES


s
-
-
---
-
-
--
CADDED
CD0


1. 6
DIAL MATIC REPRESENTATION OF THE MACROMOLECULAR STRUCTURE IN THE MITOCHONDRIAL DIVISION IN
PHYTOHA E MAGGLUTININ-STIMULATED HIMAN LYMPHOCYTES

,
.
. 요요요요요여 ​이
​,
| 01
오오오오오오오오오오오​..
M8 nosolayer
H: load
38 stalk
여여
​• H's nowly formed head
S's nowly formod stalk
|
.
.
.
.
.
…
.
. .
.
………
…
..


y
5
-
-
-
-
.
-
w
..
.
:-
This paper was submitted for publication
in the open literature at least 6 months
prior to the issuance date of this Micro-
card. Since the U.S.A.E.C. has no evi-
dence that it has been published, the pa-
per is being distributed in Microcard
form as a preprint.
DATE FILMED
16 / 29 /65





?
- LEGAL NOTICE
This roport was proparod as an account of Govoramont sponsored work. Neithor the United
slatos, nor the Commission, nor any person acting on behalf of the Commission:
A. Makos any warranty or roprosentation, oxprossod or impliod, with respoct to the accy-
racy, complotonoss, or usofulnous of the Information contained in this roport, or that the uso
of any information, apparatus, molbod, or process disclosed in this roport may not infringe
privatoly ownod rights; or
B. Assumos any liabilities with rospect to the use of, or lor dainagoy rosulung from tho
uso of any information, apparatus, method, or procon disclosod in this report.
As usod in the abovo, “porson acting on behall of the Commission" Includes any om-
ployoo or contractor of the Commission, or omployee of such contractor, to the oxtont that
such omployoo or contractor of the Commission, or employoo of such contractor proparos,
dissominatos, or provides accou, to, any Information pursuant to his omployment or contract
with the Commission, or hio omployment wilh such contractor.

END