Stephen
M. Wolniak
Professor
Department
of Cell Biology &
Molecular Genetics
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Formation
of the Cytoskeleton and Motile Apparatus
in Spermatids of Marsilea |
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University
of Maryland |
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College
Park, Maryland 20742 |
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e-mail: swolniak@umd.edu |
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Marsilea
vestita is a water
fern (Figure
1) that grows as a weed at the
edges of vernal pools or in rice paddies. By
all outward appearances, the plant resembles
a large four-leafed clover (Figure
2), but in reality, it is only
distantly related to the flowering plants.
The meiotic products from the sporophyte, the
microspores and megaspores, are found within
hardened structures known as sporocarps (Figure
3). After being placed into water,
the dry microspores develop into male gametophytes
and the megaspores develop into female gametophytes.
Fertilization in lower plants involves the
fusion of a motile male gamete, the spermatozoid,
with a non-motile female gamete that is encased
in a flask-shaped array of vegetative cells
known as an archegonium. Plant spermatozoids
are structurally complex cells (Figure
1) that differentiate within an
antheridium. The release of certain chemicals
(i.e., pheromones) from female gametes stimulate
changes in spermatozoid swimming behavior and
direction; these cues effectively guide the
spermatozoids toward the egg. This change in
swimming behavior is known as chemotaxis, a
word that was coined for the response of fern
spermatozoids to chemicals present in egg exudates
[Pfeffer, 1884].
Almost
90 years ago, Lester Sharp [1912] described the
rapid process of spermatozoid formation in the
male gametophytes of M. vestita. He found
that the placement of dry spores into water would
result in sperm formation; the process reached
completion in about eleven hours with the release
of 32 of these spirally shaped spermatozoids. The
development of the male gametophyte occurs entirely
within the microspore wall. Sharp tracked the process
microscopically and determined that during the
first 5.5 hours, there were nine mitotic divisions,
which occurred in precise division planes to produce
a total of 39 cells (Figure
4). When the division cycles are complete,
the gametophyte consists of one prothallial cell,
six sterile jacket cells and 32 spermatids. Then,
each of the 32 spermatid cells differentiates into
a motile spermatozoid. Each spermatozoid had ~140
cilia. Midway through the process, and before the
last two division cycles are complete, Sharp observed
that the appearance of a particle known as the
blepharoplast (i.e., eyelash [Webber, 1899]) preceded
sperm elongation and assembly of the cilia. Sharp
saw the blepharoplast initially as a dark dot in
the cells. In 1976, Peter Hepler provided high
resolution images of the blepharoplast (Figure
5), and demonstrated that this spherical
structure was the site of basal body synthesis.
Basal bodies serve as the templates for ciliary
and flagellar axonemes. This process is important
because in animal cells, basal bodies are formed
in close association with centrioles. Plant cells
lack centrioles. The process of blepharoplast formation
provides a unique opportunity to study basal body
formation in a cell where preexisting centrioles
are lacking, and a situation where the process
is triggered at a precise time, by an easily repeated
process (placing spores into water). Blepharoplasts
form 4 hours after the spores are placed into an
aqueous medium. During the last half of gametophyte
maturation, the development of the spermatids centers
on formation of a complex cytoskeletal array known
as a multilayered structure (MLS,
Figure 6) , the elongation and coiling
of the cell body, nuclear elongation and chromatin
packing, and finally, ciliogenesis. The structural
aspects of this complicated morphogenetic process
have been documented by Myles and Hepler (1977).
Studies
recently initiated in my laboratory have focused
on the identity of some of the proteins that are
present in the blepharoplast, and we have extended
these findings in experiments designed to test
whether the synthesis of these components is necessary
and sufficient for the formation of basal bodies. Marsilea gametophytes
provide a unique opportunity to study de novo basal
body formation in populations of synchronously
developing populations of cells. The process is
rapid and is activated by the simple addition of
water. Marsilea vestita is not a model
system in the standard sense, where genetic approaches
permit the generation of organisms exhibiting permanent
shifts in structure, development, or in physiological
capacity or response, so we have developed a variety
of alternative strategies and probes to study this
process. We have devised protocols to isolate mRNA
from these gametophytes and we have made a cDNA
library from gametophytes at all stages of development
[Hart and Wolniak, 1999]. We have devised immunolabeling
protocols for in situ detection of antigens
in cells, for identification of polypeptides isolated
from the gametophytes [Klink and Wolniak, 2001].
In parallel, we have devised protocols for the
detection of specific mRNAs in cells, by using in
situ hybridization in our plastic-embedded
sections of gametophytes [Tsai and Wolniak, 2001].
Many aspects of the technique development phase
in this project were time-consuming processes.
We performed a random screen of our cDNA library
and have now sequenced and analyzed several hundred
cDNA inserts. Approximately 50% of these cDNAs
represent known gene products that are involved
in cell cycle regulation, spindle and cytoskeletal
organization, and cell motility. Thus, we have
established a species-specific gene bank to study
spermiogenesis in this organism. We have developed
and employed a series of antisense and RNAi strategies
and technologies with Marsilea gametophytes
[Klink and Wolniak, 2000, 2001].
For
the past several years, we have focused specifically
on identifying what components are necessary for
blepharoplast formation and the subsequent assembly
of basal bodies. Our initial experiments [Hart
and Wolniak, 1998] revealed that large quantities
of components used in the formation of the motile
apparatus are stored as proteins that are present
in the dry spore (Figure
7). We did not see any distinct differences
in protein isolates from gametophytes treated with
a-amanitin (Figure
7, an inhibitor or transcription)
or cycloheximide (Figure
7, an inhibitor of translation). Prime
examples of stored proteins are the tubulins, which
represent the bulk of the fiberous portion of the
cytoskeleton and the ciliary axonemes; in both
structures, the tubulin subunits are assembled
into microtubules. New ß-tubulin is synthesized
in these cells only late in spermiogenesis, when
the spermatids are engaged in ciliary axoneme formation.
We have also found that large quantities of stored
mRNA are also present in the dry spore. This mRNA
is used for the translation of new proteins (Figure
8). Remarkably, there is little if
any transcription in the gametophyte during its
11 hour developmental cycle; development progresses
quite far in the presence of a-amanitin, a transcriptional
inhibitor (Figure
9 and Figure
10). In contrast, in the absence of
translation brought about by treatment with cycloheximide
(Figure
9 and Figure
10), there is little discernable development
of the gametophyte. Thus, development of the gametophyte
relies on the translation of stored mRNAs. One
of these proteins made from stored mRNA is centrin (Figure
11), which functions in the blepharoplast (Figure
12) and elsewhere to orchestrate patterns
and extents of macromolecular assembly of proteins
like the tubulins. We found that centrin is made
from stored mRNA (Figure
11) and that the abundance of the
protein increases dramatically at the time the
blepharoplast appears in the cells [Hart and Wolniak,
1998, 1999] (Figure
12). We isolated a cDNA from our library
that encodes a legitimate centrin that we have
named MvCen1 (Figure
13). This predicted protein compares
well with known centrins from a variety of eukaryotes (Figure
13). We developed the working hypothesis
that the timing of centrin synthesis is directly
coupled with the activity of centrin in making
blepharoplasts form.
By
employing RNAi strategies, we have found that centrin
translation is indeed necessary for the formation
of the blepharoplast [Klink and Wolniak, 2001].
Antisense centrin RNA blocks development at a stage
when the blepharoplast should be present, but blepharoplasts
are not detectable in these cells. Sense centrin-RNA
inhibits development, because of RNAi (RNA interference)
effects, as shown with dsRNA, which is inhibitory
at significantly lower concentrations (Figure
14). It is clear that we have induced
a form of post-transcriptional gene silencing,
which blocks progression in spermatid development
in a stage-specific fashion, through the targeted
destruction of centrin mRNA in the gametophyte
(Figure
15 and Figure
16). The concentration-dependence
of inhibition using centrin-derived RNA probes
strongly supports the notion that our treatments
of cells with centrin antisense RNA or centrin
dsRNA are gene-specific blocks to centrin translation,
because these probes target the destruction of
centrin mRNA (Figure
15 and Figure
16). In control experiments, we have
been able to determine that ß-tubulin antisense
RNA has no effect on development until late in
spermiogenesis, when ß-tubulin translation
is required for ciliary growth. We have been able
to show that an irrelevant RNA, a noncoding sequence
from HIV, when added to our cells as an antisense
or dsRNA construct, exerts absolutely no effect
on spermiogenesis (Figure
15 and Figure
16). Immunoblot analyses of centrin
protein production in the presence of any of these
RNA probes reveals that the block to centrin translation
only occurs when antisense or dsRNA from centin
is added to the gametophytes (Figure
14).
We
next asked if centrin translation from stored mRNA
is dependent on cell division in the gametophytes
[Tsai and Wolniak, 2001]. We blocked cell proliferation
in the gametophytes in three independent ways,
using pharmacological treatments with hydroxyurea
or olomoucine, or by RNAi treatments with dsRNA
derived from cyclin B, or dsRNA from cyclin A (Figure
17). Divisions are completely arrested
by hydroxyurea treatments administered at the time
of imbibition (Figure
17). In contrast, one or two divisions
with a few incomplete cell plates resulted from
treatments with olomoucine (Figure
17), a cyclin-dependent kinase inhibitor.
Isolomoucine was without effect on divisions or
development of the gametophytes (data not presented).
dsRNA probes for Marsilea cyclin A or cyclin B
block mitosis in the gametophyte; antisense or
dsRNA for cyclin A allow one or two divisions,
while similar probes made from cyclin B arrest
divisions completely (Figure
17). None of these treatments forestalls
or limits centrin translation, as depicted on western
blots obtained from protein isolates of cell populations
harvested at 2 h intervals during development (Figure
18), but in the absence of mitosis,
we have been unable to detect blepharoplasts. Thus,
centrin translation appears to be necessary but
not sufficient for blepharoplast formation. Cytoplasmic
partitioning appears important for organizing components
that assemble into the blepharoplast as these cells
acquire an organized centrosome and initiate de
novo basal body assembly.
Through
the largesse of active members in the cytoskeletal,
centrosomal, and axonemal research communities,
we have obtained a number of different antibodies
that bind to antigens present in our developing
spermatids. We have expanded on our immunoblot
analyses [Hart and Wolniak, 1998], looking for
changes in the abundance of some 70 antigens in
protein isolates obtained at different stages during
spermiogenesis (Klink and Wolniak, unpublished
observations). In contrast to alpha-, beta-
and gamma-tubulin, which are abundant
as proteins in the dry spores, but in a fashion
similar to centrin, the translation of p28, RanBPM
and Xgrip109 from stored mRNA precedes or accompanies
blepharoplast appearance. Thus, we have additional
candidate proteins whose synthesis parallels centrin
translation patterns; these candidates will be
the subjects for further analysis of blepharoplast
composition and formation.
We
have expanded our research interests in new directions,
asking how a single cell can rapidly
undergo a series of division cycles, and produce
two distinct kinds of cells [Tsai et al.,
2004]. The division cycles in the gametophyte ultimately
give rise to nine sterile cells and 32 spermatids.
Two clusters of 16 spermatids are formed from the
two spermatogenous initials, which differ compositionally
and structurally from the adjacent sterile jacket
cells that surround them. When we began to look at
patterns of stored protein accumulation, and new
translation, it was not particularly surprising to
find high levels of stored tubulin and new centrin
protein in the spermatogenous cells, but largely
absent from the sterile cells. We began to look at
the movement and distribution of proteins (with immunolabeling
assays) and stored mRNAs (with in situ hybridization
assays) into the cellular domains that would later
become spermatogenous initials. Surprisingly, the
distribution of most stored mRNAs does not match
that of the proteins (Figure
19 and Figure
20). Most
of the mRNAs are present in all of the cells of the
gametophyte
(Figures
19,
20,
21), while the stored proteins
like ß-tubulin
(Figure
19) or newly translated proteins like
centrin (Figure
20) become exclusively localized
in the spermatogenous
initials. We were surprised to find that cyclin B
mRNA was present in all cells of the gametophyte
(Figure 21), though the jacket cells, once formed,
cease to divide. We were able to find several mRNAs
that exhibit nonrandom distributions in spermatogenous
cells; two of these transcripts encode a PRP-19 like
protein and an RNA helicase (Figure
22). RNAi experiments
using dsRNAs made from either of these transcripts
results in anomalous division patterns in the gametophyte
(Figure
22). We found that patterns of polyadenylation
match the distribution of transcription patterns,
mostly in spermatogenous cells (Figure
23), and we
also showed that the cytoplasmic polyA polymerase
(PAP) protein is present and abundant in spermatogenous
cells, but PAP is barely detectable in sterile jacket
cells (Figure
24). These results suggest that translational
capacity in the spermatogenous cells is regulated
at several levels, and some of this control apparently
involves mRNA processing.
In ongoing work, we have used these mRNAs in assays
to study transcript movement prior to cell partitioning
within the fixed volume of the gametophyte. These
experiments provide insights on cell fate determination
in plants, a process that is tightly coupled with
cell position and cell size in tissues and organs
where cell movements do not occur.
Hart,
P. and S.M. Wolniak. 1998. Spermiogenesis
in Marsilea vestita: a temporal correlation
between centrin expression and blepharoplast differentiation. Cell
Motil. Cytoskelet. 41: 39-48.
[Cover Illustration 42(2)]
Hardin,
S. and S.M. Wolniak. 1998. Molecular cloning and
characterization of maize ZmMEK1, a protein kinase
with a catalytic domain homologous to mitogen-
and stress-activated protein kinase kinases. Planta 206: 577-584.
Hart,
P. and S.M. Wolniak. 1999. Molecular
cloning of a centrin homolog from Marsilea
vestita and evidence for its translational
control during spermiogenesis. Biochem.
Cell Biol. 77: 101-108.
Wolniak,
S.M., V.P. Klink, P.E. Hart and C.-W. Tsai. 2000.
Control of the development and motility in the
spermatozoids of lower plants. Grav. Sp. Biol.
Bull. 13: 85-93.
Klink,
V.P. and S.M. Wolniak. 2001. The
efficacy of RNAi in the study of the plant cytoskeleton. J.
Pl. Growth Reg. 19: 371-384.
Klink,
V.P. and S.M. Wolniak. 2001. Centrin
is necessary for the formation of the motile apparatus
in spermatids of Marsilea. Mol.
Biol. Cell 12: 761-776.
Hardin,
S. and S.M. Wolniak. 2001. Expression of the mitogen-activated
proteinkinasekinase ZmMEK1 in the primary root
of maize. Planta 213: 916-926.
Tsai,
C.W. and Wolniak. S.M. 2001. Cell
cycle arrest allows centrin translation but not
basal body formation during spermiogenesis in Marsilea. J.
Cell Sci. 114: 4265-4272 [Cover
Illustration 114(23)].
Klink, V.P. and .M. Wolniak. 2003. Changes in the
abundance and distribution of conserved centrosomal,
cytoskeletal and ciliary proteins during spermiogenesis
in Marsilea vestita. Cell Motil. Cytoskelet. 56:
57-73.
Tsai, C.W., C.M. Van der Weele and S.M. Wolniak.
2004. Differential segregation and modification of
mRNA during spermiogenesis in Marsilea vestita. Develop.
Biol. 269: 319-330.
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