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developmental
checkpoints
Checkpoint
Controls
Cellular differentiation
in all organisms requires a defined program of physiological and
morphological changes that result in the formation of an alternate
cell-type. Such structures range from a bacterial flagellum, to
a dormant life form such as a bacterial endospore or cyst, or
chorion assembly in an insect. Orchestrating such changes in a
cell is an underlying program of gene expression exquisitely co-ordinated
over a period of time by the incorporation of a number of regulatory
factors that govern and modulate differential gene expression.
Successful
completion of any program of cell differentiation requires accurate
synthesis and assembly of macromolecules within the cell. As the
complexity and time required for such tasks increases so does
the likelihood that erroneous synthesis and assembly might arise
leading to the formation of aberrant macromolecules or structures.
To ensure a high fidelity in the synthesis and assembly of biological
structures the cell has developed a feedback mechanism where certain
'landmark' events that arise during development can be used to
couple the program of differential gene expression with the attainment
of that 'landmark' event. For example, in Salmonella expression
of the genes required for the final stages of flagellum synthesis
are coupled to the construction of a complete basal body-hook
structure (Losick & Shapiro 1993). This is achieved by ensuring
that the transcription factor (sigma-28) responsible for late
fla gene expression is inactivated by the anti-sigma factor,
FlgM. However, as soon as the basal body-hook is fully built FlgM
is actually secreted through the hook (which serves as a protein-export
apparatus), thus lowering the cellular concentration of anti-sigma
factor, releasing the inhibition on sigma-28 and ensuring a rapid
burst of late gene expression, completing flagellum biosynthesis.
This elegant control is termed a checkpoint and represents a mechanism
for enforcing a dependency of events to a biological process.
Thus, completion of a landmark event (completion of basal body-hook
structure) is used as a control to ensure that 'late' events under
the control of sigma-28 can only proceed after completion of 'early'
events. In the absence of such a control, aberrant flagellum synthesis
occurs. Ironically, some intact functional flagella are produced
in the absence of the checkpoint but these are the few that manage
to complete the complex program of flagellum synthesis by chance,
therefore a checkpoint ensures the high fidelity of a morphological
process.
In the eukaryotic
cell division cycle where a well defined sequence of events is
required for the high fidelity of chromosome transmission, the
absence of an ordered sequence of events can lead to abnormal
growth or even cell death. Not surprisingly, a number of crucial
events in the cell division cycle incorporate checkpoint controls,'
e.g., the dependence of mitosis on completion of DNA replication
(the RAD9 checkpoint) and the dependence of anaphase on microtubule
spindle formation ( Enoch & Nurse, 1991; Hartwell & Weinert,
1989; Li & Murray, 1991; Weinert & Hartwell, 1993). It
would seem highly probable that such checkpoint controls are used
universally in processes ranging from the formation of a spore-forming
bacterium to development of the vertebrate embryo. Such control
mechanisms provides important insights into understanding how
a complex biological process can be accurately coordinated over
a period of time. Intriguingly, in tumour formation, where cell
growth is no longer controlled it is likely that such checkpoints
in the cell's division cycle have been bypassed leading to aberrant
cell growth.
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Checkpoints
in Bacillus subtilis
The prokaryote
Bacillus subtilis has provided a model example of unicellular
differentiation, which because of the excellent genetics available
has provided an excellent system to understand the basic mechanisms
of regulating developmental gene expression. As a response to
nutrient exhaustion B. subtilis enters a program
of irreversible differentiation that results in the formation
of a dormant life form termed the endospore or spore ( Errington,
1993; Piggot & Coote, 1976 ). This process lasts some 8 hours
and uses approximately 70 developmental genes ( Errington, 1993;
Stragier, 1994 ). A number of well defined physiological and morphological
changes occur during this process but the landmark event is the
formation of two compartments within the developing cell or sporangium,
termed the forespore and mother cell. The forespore is the germ
line cell destined to become the mature spore but is made first
by a process of membrane invagination which encases one of the
two chromosomes present in the developing cell in two layers of
phospholipid membrane. In both spore chambers separate programs
of temporal gene expression occur, each driven by RNA polymerase
bound to one of five alternate sigma factors ( Stragier &
Losick, 1990). These transcription factors appear at different
times during development and, by recognizing different promoters,
ensure expression of a unique regulon of genes during development.
Four of these sigma factors are active only in the forespore or
mother cell chambers providing spatial control of gene expression
as well as temporal control. In addition, a number of DNA-binding
proteins can further modulate gene expression.
At least three
checkpoints controls are
utilized during sporulation which control the activity of the
transcription factors sigma-E,-G and-K. In each case the sigma
factor is synthesised first in an inactive form and is rendered
active only after the cell has reached some critical point in
development (Losick & Stragier, 1992). The mother cell transcription
factors sigma-E and sigma-K both require proteolytic cleavage
of a short N-terminal leader sequence to be rendered active. In
both the sigma-E and sigma-K -checkpoints, the putative protease
that cleaves the sigma factor, and the signalling molecule that
activates processing has been identified. Interestingly, in both
examples the checkpoint requires the delivery of an intercompartmental
signal to stimulate processing of the sigma factor. Thus, pro-sigma-E
synthesized in the mother cell at stage II requires a signal from
the opposed prespore chamber to facilitate processing of pro-sigma-E.
Both the signal molecule (SpoIIR) and putative protease (SpoIIGA)
have been identified (Karow et al., 1995; Londono-Vallejo
& Stragier, 1995; Stragier et al., 1988). Pro-sigma-K
is synthesized at stage III in development in the mother cell
and requires an intercompartmental signal from the forespore chamber
to activate processing of pro-sigma-K (Cutting et al., 1990).
Again, the putative protease (SpoIVFB) and signalling molecule
(SpoIVB) have been identified ( Cutting et al., 1991a;
Cutting et al., 1991b ). In both checkpoints, late events
under the control of sigma-E or sigma-K are dependent upon completion
of early events which lead to signal production. In the sigma-K
checkpoint, removal of the checkpoint results in the production
of aberrantly formed spores, which although viable, are unable
to survive the adverse conditions tolerated by wild-type spores.
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Identification
of the Sigma K Checkpoint
The sigma-K
checkpoint was first identified because of the dependence of mother
cell sigma-K -directed gene expression upon forespore gene expression
under the control of sigma-G (Cutting et al., 1990). Since
gene expression controlled by these two transcription factors
was separated by the two membranes of the forespore we speculated
that some event under sigma-G control was required for activation
of sigma-K in the opposed mother cell chamber. It has-been demonstrated
that sigma-K is first made as an inactive proprotein, pro-sigma-K
which is activated by proteolytic cleavage of a 20 aa. N-terminal
fragment (Lu et al., 1990). In its mature form sigma-K
can proceeds to direct the expression of a regulon of genes that
are involved in the terminal stages of spore development, in particular,
the synthesis and assembly of the spore coat ( the protective
proteinaceous barrier that provides the spore with its resistant
properties). Pro-sigma-K is made at stage III in sporulating cells
yet must wait and accumulate for 1 hour before activation, thus
the checkpoint provides a control ensuring that sigma-K is only
activated at a precise point in development. We have identified
the principal components of this checkpoint as summarized below.
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The
Putative Protease
The gene
products required for processing pro-sigma-K to its mature form
comprise at least three polypeptides, SpoIVFA, SpoIVFB and BofA.
These molecules are thought to form a heteroligomeric complex in the outermost layers
of the forespore where they somehow facilitate cleavage of pro-sigma
-K in the mother cell. SpoIVFB has been shown from genetic and
biochemical experiments to be the most likely candidate for the
processing enzyme that cleaves pro-sigma-K ( Cutting et al.,
1991b; Lu et al., 1995 ). SpoIVFA and BofA are inhibitors
of SpoIVFB, such that in their absence processing of pro-sigma-K
is not controlled thus uncoupling the requirement of mother cell
gene expression on forespore events ( Cutting et al., 1991b;
Ricca et al., 1992).
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The
signal
The one gene
product required for signalling processing of pro-sigma-K is the
46 kDa. SpoIVB protein ( Cutting et al., 1991a ). This
protein is synthesized in the forespore chamber (the spoIVB
gene is sigma-G -dependent) and we have shown that SpoIVB is the
only forespore gene essential to activate processing of pro-sigma-K
SpoIVB contains an N -terminus similar to those of lipoproteins
which may suggest that it can transverse the inner membrane of
the forespore putting in a position where it is in close proximity
to the SpoIVFA/BofA/SpoIVFB complex in the outer forespore membranes
(Gomez et al., 1995; Oke et al., 1997). Lastly,
we have identified the BofC protein that may function as a chaperone-like
molecule facilitating SpoIVB's signalling role (Gomez & Cutting,
1997).
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The
Timing Mechanism
We have recently
discovered a gene, spoVT, whose product is a transcriptional
regulator of the spoIVB gene (Bagyan et al., 1996). Similar
to the AbrB DNA-binding protein SpoVT serves as an enhancer of
spoIVB sigma -G -controlled expression. In SpoVT's absence
spoIVB expression is delayed which causes a 90 minute delay
in signal transduction and processing of pro-sigma-K. We believe
that SpoIVB must reach critical threshold level in the forespore
before its is able to activate the SpoIVFB/SpoIVFA/BofA processing
complex. SpoVT ensures that this threshold level is reached at
the critical time in development.
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References
Bagyan,
I., Hobot, J. & Cutting, S. M. (1996). A compartmentalized
regulator of developmental gene expression in Bacillus subtilis.
J Bacteriol 178, 4500-4507.
Cutting,
S., Driks, A., Schmidt, R., Kunkel, B. & Losick, R. (1991a).
Forespore-specific transcription of a gene in the signal transduction
pathway that governs pro-sigma-K processing in Bacillus subtilis.Genes
Develop 5, 456-466.
Cutting,
S., Oke, V., Driks, A., Losick, R., Lu, S. & Kroos, L. ( 1990
). A forespore checkpoint for mother cell gene expression
during development in B. subtilis. Cell 62,
239-250.
Cutting,
S., Roels, S. & Losick, R. (1991b). Sporulation operon
spoIVF and the characterization of mutations that uncouple
mother-cell from forespore gene expression in Bacillus subtilis.
J Mol Biol 221, 1237-1256.
Enoch,
T. & Nurse, P. ( 1991 ). Coupling M phase and S phase
: controls maintaining the dependence of mitosis on chromosome
replication. Cell 65, 921-923.
Errington,
J. (1993). Bacillus subtilis sporulation : regulation
of gene expression and control of morphogenesis. Microbiol
Rev 57, 1-33.
Gomez,
M. & Cutting, S. (1997). bofC encodes a putative
forespore regulator of the Bacillus subtilis sigma-K checkpoint.
Microbiol
Gomez,
M., Cutting, S. & Stragier, P. (1995). Transcription of
spoIVB is the only role of sigma-G that is essential for
pro-sigma-K processing during spore formation in Bacillus subtilis.
J. Bacteriol 177, 4825-4827.
Hartwell,
H. & Weinert, T. A. (1989). Checkpoints: controls that
ensure the order of cell cycle events. Science 246,
629-634.
Karow,
M. L., Glaser, P. & Piggot, P. J. (1995). Identification
of a gene, spoIIR, which links activation of sigma-E to
the transcriptional activity of sigma-F during sporulation in
Bacillus subtilis. PNAS USA 92, 2012-2016.
Lif R.
& Muffay, A. W. (1991). Feedback control of mitosis in
budding yeast. Cell 66, 519-531.
Londono-Vallejo,
J.-A. & Stragier, P. ( 1995 ). Cell-cell signalling pathway
activating a developmental transcription factor in Bacillus
subtilis. Genes Develop 9, 503-508.
Losick,
R. & Shapiro, L. (1993). Checkpoints that couple gene
expression to morphogenesis. Science 262, 1227-1228.
Losick,
R. & Stragier, P. ( 1992 ). Crisscross regulation of cell-type
-specific gene expression during development in B. subtilis.
Nature 355, 601-604.
Lu, S.,
Cutting, S. & Kroos, L. ( 1995 ). Sporulation protein
SpoIVFB from Bacillus subtilis enhances processing of the
sigma factor precursor pro-sigma-K in the absence of other sporulation
gene products. J. Bacteriol 177, 1082-1085.
Lu, S.,
Halberg, R. & Kroos, L. (1990). Processing of the mother-cell
sigma factor, sigma-K, may depend on events occuring in the forespore
during Bacillus subtilis development. Proc Natl Acad
Sci USA 87, 9722-9726.
Oke, V.,
Shchepetov, M. & Cutting, S. (1997). SpoIVB has two distinct
functions during spore formation in Bacillus subtilis.
Mol Microbiol
Piggot,
P. J. & Coote, J. G. (1976). Genetic aspects of bacterial
endospore formation. Bacteriol Rev 40, 908-962.
Ricca,
E., Cutting, S. & Losick, R. (1992). Characterization
of bofA, a gene involved in intercompartmental regulation
of pro-sigma-K processing during sporulation in Bacillus subtilis.
J Bacteriol 174, 3177-3184.
Stragier,
P. (1994). A few good genes : developmental loci in Bacillus
subtilis.In Spores XI: Regulation of Bacterial Differentiation.,
pp. 207-245. Edited by Piggot., P. J., Youngman., P. and Jr.,
C. P. M. Washington, D.C.: American Society for Microbiology.
Stragier,
P., Bonamy, C. & Kar.Tnazyn-Campbelli, C. (1988). Processing
of a sporulation sigma factor in Bacillus subtilis: how
morphological structure could control gene expression. Cell
52, 697-704.
Stragier,
P. & Losick, R. (1990). Cascades of sigma factors revisited.
Mol Microbiol 4, 1801-1806.
Weinert,
T. A. & Hartwell, L. H. (1993). Cell cycle arrest of cdc
mutants and specificity of the RAD9 checkpoint. Genetics
134, 63-80.
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