evidence for an intracellular precipitated, noncrystalline Ca precursor that is shuttled along the syncytial cytoplasmic strand and secreted into the syncytium in
which the spicule forms.
7.2.3 Occluded Proteins
Spicule matrix proteins are also synthesized and secreted by PMCs; thereafter, the
proteins can be found occluded within the mineral phase. The earlier literature on
this subject can be found in the review of Decker and Lennarz (1988). There has
been very little work done on the synthesis, delivery, and occlusion of these
proteins during spicule formation. Benson et al. (1989) demonstrated, using polyclonal antibodies against an undefined mixture of matrix proteins from the spicule,
that intracellular vesicles, which contained putative matrix proteins, could be
identified in PMCs. The first matrix proteins studied by modern methods, SM50
and SM30, have been the subject of several studies. Ingersoll et al. (2003) using
immunoelectron microscopy and affinity-purified antibodies showed that both of
these proteins are found in the Golgi apparatus and in transport vesicles (approximately 50 nm in size), that deliver their contents to the space in which the spicule is
formed. Wilt et al. (2008b) followed GFP-tagged versions of SM30 and SM50; in
both instances, the PMCs harboring the transgene secreted GFP-tagged SM30/50
into the spicule space near the PMC of origin. This finding contrasts with the
delivery of calcium, which shuttles primarily to the extending tip. The kinetics of
secretion of
35 S-labeled SM30 were followed in PMCs by immunoprecipitation of
the protein in the cell and spicule, formed in culture. It took about 20 min after the
introduction of label for secreted proteins to begin accumulation in the forming
spicule, and the SM30 delivered to the spicule had a somewhat lower molecular
mass, indicating that processing of the protein had occurred during the secretion
and/or spicule assembly steps.
There are a variety of studies that characterize the cell biology of spicule formation. Inhibitors of metalloproteases stop spicule elongation (Ingersoll and Wilt 1998;
Huggins and Lennarz 2001), though this inhibition does not interfere with initial
formation of the calcite rhombohedra in the PMCs. It is no surprise that inhibitors of
ion transport have a deleterious effect (e.g., Mitsunaga et al. 1986). The mechanism
(s) by which vectorial secretion of matrix proteins is accomplished is unknown. We
do know that PMCs secrete many other molecules, including collagens and
proteoglycans, into the blastocoel (Benson et al. 1990), but such molecules are not
found occluded in the spicule, nor are spicule proteins found in the blastocoel.
Finally, it is important to remember that spicule formation may only represent
one mode of biomineralization. The biomineral is not assembled on a scaffold at
some distance from the cell, as is the case in mollusks, or vertebrate bone, but in
intimate contact with the cell. The spicule is completely enrobed by syncytial
204
P.U.P.A Gilbert and F.H. Wilt
which the spicule forms.
7.2.3 Occluded Proteins
Spicule matrix proteins are also synthesized and secreted by PMCs; thereafter, the
proteins can be found occluded within the mineral phase. The earlier literature on
this subject can be found in the review of Decker and Lennarz (1988). There has
been very little work done on the synthesis, delivery, and occlusion of these
proteins during spicule formation. Benson et al. (1989) demonstrated, using polyclonal antibodies against an undefined mixture of matrix proteins from the spicule,
that intracellular vesicles, which contained putative matrix proteins, could be
identified in PMCs. The first matrix proteins studied by modern methods, SM50
and SM30, have been the subject of several studies. Ingersoll et al. (2003) using
immunoelectron microscopy and affinity-purified antibodies showed that both of
these proteins are found in the Golgi apparatus and in transport vesicles (approximately 50 nm in size), that deliver their contents to the space in which the spicule is
formed. Wilt et al. (2008b) followed GFP-tagged versions of SM30 and SM50; in
both instances, the PMCs harboring the transgene secreted GFP-tagged SM30/50
into the spicule space near the PMC of origin. This finding contrasts with the
delivery of calcium, which shuttles primarily to the extending tip. The kinetics of
secretion of
35 S-labeled SM30 were followed in PMCs by immunoprecipitation of
the protein in the cell and spicule, formed in culture. It took about 20 min after the
introduction of label for secreted proteins to begin accumulation in the forming
spicule, and the SM30 delivered to the spicule had a somewhat lower molecular
mass, indicating that processing of the protein had occurred during the secretion
and/or spicule assembly steps.
There are a variety of studies that characterize the cell biology of spicule formation. Inhibitors of metalloproteases stop spicule elongation (Ingersoll and Wilt 1998;
Huggins and Lennarz 2001), though this inhibition does not interfere with initial
formation of the calcite rhombohedra in the PMCs. It is no surprise that inhibitors of
ion transport have a deleterious effect (e.g., Mitsunaga et al. 1986). The mechanism
(s) by which vectorial secretion of matrix proteins is accomplished is unknown. We
do know that PMCs secrete many other molecules, including collagens and
proteoglycans, into the blastocoel (Benson et al. 1990), but such molecules are not
found occluded in the spicule, nor are spicule proteins found in the blastocoel.
Finally, it is important to remember that spicule formation may only represent
one mode of biomineralization. The biomineral is not assembled on a scaffold at
some distance from the cell, as is the case in mollusks, or vertebrate bone, but in
intimate contact with the cell. The spicule is completely enrobed by syncytial
204
P.U.P.A Gilbert and F.H. Wilt