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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 4004-4007
BRIEF REPORT
Megakaryocyte dense granule components are sorted in
multivesicular bodies
Tayebeh Youssefian and
Elisabeth M. Cramer
From the INSERM U 474, Hôpital Cochin-Port-Royal, Paris,
France.
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Abstract |
Recent studies suggest that multivesicular bodies are an
intermediate stage in the formation of  -granules. In contrast, the kinetics and mode of appearance of dense granules during megakaryocytic maturation has remained poorly understood. Immunoelectron microscopy was used to monitor the appearance of dense granular markers
(granulophysin and serotonin) on cryosections of human megakaryocytes
(MKs) cultured from CD34+ precursors. The monitoring was
done on days 8 and 13 of culture. The data suggest that dense granules
appear in immature MKs early during their maturation, concomitantly
with -granule formation. In MKs of intermediary maturation stage,
granulophysin was mainly localized within dense granules and
multivesicular bodies (MVBs), which were also labeled for serotonin.
This study provides evidence that granulophysin is a dense granule
marker in human MKs and that MVBs are an intermediary stage of dense
granule maturation and probably constitute a sorting compartment
between -granules and dense granules.
(Blood. 2000;95:4004-4007)
© 2000 by The American Society of Hematology.
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Introduction |
Dense granules belong to the secretory compartment that
is released during platelet activation. They contain a variety of important hemostatically active substances1 including
matrix proteins that are released into the extracellular medium upon activation (eg, serotonin, catecholamines, adenosine
5'-diphosphate [ADP], adenosine 5'-triphosphate [ATP],
and calcium) and integral components2 of the granule
limiting membrane (eg,  IIb 3; glycoprotein Ib [GPIb];
P-selectin2,3; and granulophysin,2,4,5 a
specific dense granule marker). However, the biogenesis of dense granules in the platelet precursor, the megakaryocyte (MK), is poorly
understood. Recently Heijnen et al6 have shown that multivesicular bodies (MVBs) represent a developmental stage in -granule maturation. Some receptors, such as IIb3, GPIb, and P-selectin, have been identified in the limiting membrane in both -granules and dense granules.2-4,7-9 In addition, these
granules share remarkable similarities in their protein trafficking
pathways, with both endogenous synthesis and endocytosis from the
extracellular medium.10-13 In this study we investigated
the formation of dense granules during MK maturation on human culture
MKs via immunoelectron microscopy. Our data indicate that MVBs are an
intermediary compartment where -granule and dense granule components
are sorted.
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Study design |
Cells
Human MKs were grown in liquid culture from bone marrow
precursors obtained from normal adult graft donors as
previously described.8
Antibodies
We used the following rabbit polyclonal antibodies (pAbs) at 10 µg/mL: antiglycocalicin, an antibody to
GPIb9,14 (gift from Dr Michael Berndt, Victoria,
Australia); antiserotonin (Sigma, St Louis, MO) and anti-von
Willebrand factor (Dako, Glostrup, Denmark).
Antigranulophysin,2 a monoclonal antibody (mAb) (gift from
Dr Israels, University of Manitoba, Winnipeg, Manitoba, Canada) was
also used at 10 µg/mL. Goat antirabbit (GAR) and antimouse (GAM)
immunoglobulin G (IgG) fractions were coupled to 5-nm or 10-nm gold
particles (British Biocell International, Cardiff, England).
Electron microscopy
MKs were prepared for immunoelectron microscopy as previously
described.3 Briefly, they were fixed in 1% glutaraldehyde, embedded in sucrose, and frozen in liquid nitrogen.15
Simple and double immunogold labeling was performed on ultrathin
cryosections.3 In parallel, control sections were incubated
with gold conjugated antibodies after omitting the primary antibodies,
which displayed no labeling. Samples were observed on a Philips CM10
electron microscope (Philips, Eindhoven, The Netherlands).
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Results and discussion |
We documented the time of appearance of dense granules during MK
maturation using immunolabeling experiments on frozen thin sections of
human MKs at day 8 of culture. Double immunolabeling for GpIb (a
specific MK marker) and granulophysin (a specific dense granule marker)
facilitated the detection of dense granules within immature MKs. Some
poorly differentiated blastic cells deprived of specific organelles
were identified as immature MKs because they displayed the
classical membrane distribution for GPIb. However, consistent
labeling for granulophysin was observed within electron
luscent granules and vacuolar-like structures similar to
the classical dense granular structure (Figure
1). One of the problems during preparation
of cryosections is that the content of dense granules often
becomes extracted, and thus the dense granules appear as vacuolar
organelles.2,3 Because of difficulty visualizing dense
granules, the time and mode of their appearance during MK maturation
has remained poorly understood. Previous investigations had shown
the presence of dense granules in MKs by using mepacrine labeling and
uranaffin cytochemical reactions, but only mature MKs had been
investigated.16 Our present experiments have focused upon
the appearance of dense granules in the early stage of MK
maturation.
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| Fig 1.
Detection of dense granules in immature MKs
at day 8 of culture by double labeling for GPIb and granulophysin.
Arrowheads indicate GPIb (15-nm gold particles), and arrows indicate
granulophysin (10-nm gold particles). This immature MK, seen on a
frozen thin section, displays a nucleus (N) with abundant euchromatin
and a prominent nucleolus (n) and appears morphologically
undifferentiated. It can only be identified by GPIb-specific expression
on its plasma membrane (pm). Granulophysin labels the membrane of
vacuolar structures, which can be identified as dense granules (d)
because of the presence of this specific marker (original magnification
×41 800; inset, ×3920).
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To document the intracellular pathway of dense granule formation,
double-labeling experiments were performed in mature MKs at day 13 of
culture (Figure 2A) using a mixture of
antigranulophysin mAb and antiserotonin pAb. Serotonin and
granulophysin were detected in some vacuolar structures, with or
without a dense content, thereby confirming the identification of these
organelles as dense granules (Figure 2B). Granulophysin appeared to be
located along the limiting membrane of these organelles as well as
around the dense vesicles which often constituted their dense core.
This result is consistent with the previous observation of serotonin uptake in the MK precursors, the megakaryoblasts.1 Both
markers were also found in MVBs (Figure 2C), and granulophysin was
mainly located on the membrane of their internal vesicles. Simple
labeling for granulophysin showed that 17% of labeling was associated
with dense granule membranes of mature Mks (Figure 2D). Approximately 80% of the labeling was found in the internal vesicles of MVBs or with
the membrane of some intermediary MVBs that had lost part of their
vesicle structure, and less than 3% of the labeling was associated
with -granules (Figure 2D, E). In addition, the granulophysin containing -granules was often immature, with a large size and odd
shape, and was localized in the vicinity of MVBs, suggesting that they
may originate from MVBs, as described by Heijnen et al.6
Mature -granules with a dense nucleoid were deprived of any
labeling.
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| Fig 2.
Frozen sections of maturing MKs,
immunolabeled for the detection of dense granule components.
(A) Frozen culture MKs can be identified by their large size, indented
nucleus (N), numerous secretory granules (g), and prominent demarcation
membrane system (dm). Numerous organelles (about 40), which were
identified as MVBs by electron microscopy at a higher magnification,
are present in this section. Arrowheads indicate dense granules (at
least 5), which were morphologically identifiable (original
magnification ×2640). (B, C) Double labeling for the 2 dense
granule markers is depicted. Arrowheads indicate
granulophysin (20-nm gold particles), and arrows indicate serotonin
(10-nm gold particles). (B) Serotonin is detected within the (C) MVBs
and (D) dense granules. Both structures are also labeled for
granulophysin (original magnification ×49 450). (D, E) Simple
immunogold labeling for granulophysin. (D) Granulophysin (arrowheads)
labels the dense granule limiting membranes (d), which appear like
vacuolar structures that represent 17% of total labeling. Internal
vesicles of MVBs represented 80% of the total labeling. Minimal
labeling (approximately 3%) was associated with immature -granules
of large size and odd shape (iA), whereas mature -granules (A) were
deprived of labeling. Go indicates the Golgi complex (original
magnification ×46 300). (E) Granulophysin in mature MKs is
mainly found within the dense granules (d). Labeling is also present on
some intermediate MVBs (im), which have lost part of their vesicles and
have an ultrastructure similar to that of dense granules (original
magnification ×46 300).
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Finally, double labeling experiments showed that both -granule
proteins (von Willebrand factor) and a dense granule marker (granulophysin) could be found in the same MVB. Moreover,
immunolabeling for granulophysin was located in internal vesicles
distinct from the ones containing von Willebrand factor within the same
MVB. The immunolabeling was also often associated with the limiting membrane of MVBs, which suggests that the various granule constituents had started separating (Figure 3). Thus,
our observations indicate that dense granule components are segregated
from other secretion granules within the MVBs and that these eventually
develop into dense granules.
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| Fig 3.
Double immunolabeling for the dense granule marker
granulophysin and the -granule marker von Willebrand factor.
Arrowheads indicate granulophysin (10-nm gold particles), and arrows
indicate -granule marker von Willebrand factor (5-nm gold
particles). Both proteins can be found within the same MVB, but they
are segregated from each other in distinct microvesicles. Granulophysin
is also preferentially associated with the limiting membrane of MVBs.
Von Willebrand factor is located in -granules (A) as well as in MVB
microvesicles (original magnification ×67 300).
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The present study was undertaken because there is cumulating evidence
demonstrating similarities between the 2 types of secretory granules,
-granules and dense granules. The plasma membrane receptor IIb3 is a component of the -granule membrane as well as the dense granule membrane.3,8 P-selectin is also specifically associated with the limiting membrane of both -granules and dense granules.2,7 The respective composition of both granule
types may reflect the dual mechanisms responsible for their protein trafficking: In addition to the classical endogenous MK
synthesis,17 -granules can also acquire their content by
incorporation of circulating plasma components.10,11
Similarly, dense granules are also able to take up serotonin from the
circulating plasma, and this phenomenon applies to platelets and
MKs.1,18 This close relationship is further evidenced by
the existence of congenital platelet disorders, such as
- storage pool disease in humans and gunmetal mutation in
mice,19 which involve the association of -granule
abnormalities with dense granule deficiency.20 In addition,
it was demonstrated that MVBs are an intermediary stage in the
formation of -granules.6 In this study we show that
dense granules follow a similar pathway.
Finally, it has also been demonstrated that through its
specific receptors, serotonin accumulation also occurs in platelets. Intracellular trafficking of serotonin receptors may resemble that of
granulophysin. First, these receptors would be present on the membrane
of the internal vesicles located within MVBs; subsequently, during
maturation, they are incorporated into the dense granule limiting
membrane. Therefore, in platelets, serotonin accumulation may occur as
a result of its specific receptors that are present in the dense
granule membrane.
In conclusion, the present study provides evidence that dense granules
are formed at the early stage of MK maturation. The study also
indicates that MVBs are a sorting compartment for -granule and dense
granule formation, where granulophysin is directed into the dense
granule compartment during MK maturation.
| |
Acknowledgments |
The authors gratefully acknowledge Dr Najet Debili for
megakaryocyte cultures, Mme Josette Guichard for laboratory help, Mr Jean-Marc Massé for photographic work, Dr Paul Harrison and Mr Alain Schmitt for improving the manuscript, and Dr Paul-Henri Roméo for his constant support.
| |
Footnotes |
Submitted October 6, 1999; accepted January 19, 2000.
T.Y. is a fellowship recipient from L'Association Nationale
pour la Recherche contre le SIDA.
Reprints: Elisabeth Cramer, INSERM U 474, Hôpital
Cochin-Port-Royal, 123 Boulevard Port-Royal, 75014 Paris, France; e-mail: elisabeth.cramer{at}cochin.inserm.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction