Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain1
Department of Biochemistry, Comenius University, Faculty of Natural Sciences, Mlynská dolina CH-1, 842 15 Bratislava, Slovakia2
Departamento de Biología y Genética. Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007, Salamanca, Spain3
Author for correspondence: María Molina. Tel: +34 91 3941748. Fax: +34 91 3941745. e-mail: molmifa{at}eucmax.sim.ucm.es
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ABSTRACT |
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Keywords: GFP, morphogenesis, Saccharomyces cerevisiae
Abbreviations: APC, anaphase-promoting complex; CDK, cell-cycle-dependent kinase
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INTRODUCTION |
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The septin-based ring at the bud neck in yeast has been found to be responsible for crucial events in cellular morphogenesis throughout the mitotic cycle, such as (a) the selection of cell polarity by interaction with the axial pole markers Bud3, Bud4 and Bud10/Axl2 (Chant et al., 1995 ; Sanders & Herskowitz, 1996
; Halme et al., 1996
; Roemer et al., 1996
); (b) chitin deposition at the bud neck via interaction with Bni4 and Chs4 (De Marini et al., 1997
); (c) a morphogenesis-dependent cell cycle checkpoint which involves protein kinases of the Nim1 family (Barral et al., 1999
; Shulewitz et al., 1999
; McMillan et al., 1999
); and (d) the spatial localization of the septation machinery at cytokinesis (Lippincott & Li, 1998
; Bi et al., 1998
; Cid et al., 1998a
). In turn, the highly dynamic cortical cytoskeleton, formed by fast-moving patches consisting of actin and a complex network of actin-binding proteins (see Botstein et al., 1997
for a review), is responsible for the direction of polarized secretion that enables bud growth. The data currently available on the function of the actin and septin cytoskeletal structures indicate that their rearrangements throughout the mitotic cycle direct bud emergence, bud growth and cytokinesis. This assumption implies that their dynamics must be accurately regulated by cell cycle controls so that synchronicity between nuclear and cortical events will allow a successful round of cell division.
Indeed, the actin cytoskeleton has been reported to be time-regulated by the turnover of G1 and B cyclins throughout the cell cycle (Lew & Reed, 1993 ), leading to the conclusion that, either directly or indirectly, the cell-cycle-dependent kinase (CDK) Cdc28 drives actin rearrangements for proper morphogenesis. Since the septin cytoskeleton is an apparently rigid structure that remains at the bud neck throughout the whole budding cycle, it has been recently hypothesized to work as a physical submembrane barrier that compartmentalizes the budding cell into a morphogenetically active daughter side and a morphogenetically inactive mother side (Barral et al., 2000
). However, the relationship between septin filaments assembly and maintenance and cell cycle regulatory mechanisms is still poorly understood. In this work we study the cell cycle landmarks that monitor the assembly of the septin ring, its maintenance and its duplication at the end of the cell cycle. We find that septin dynamics are driven by cyclin/CDK-dependent functions and that they are constant under different developmental programmes, such as zygotic bud growth and pseudohyphal differentiation.
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METHODS |
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Western blotting was performed as follows: 20 ml aliquots of cell culture were placed on ice in a Falcon centrifuge tube and pelleted in a refrigerated centrifuge. Cells were then resuspended in 1 ml ice-cold water and transferred to an Eppendorf tube, pelleted and immediately frozen on dry ice. Cells were broken in 120 µl cold lysis buffer [50 mM Tris/HCl (pH 7·5), 10% glycerol, 1% Triton X-100, 0·1% SDS, 150 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 50 mM ß-glycerol phosphate, 5 mM sodium pyrophosphate, 5 mM EDTA, 1 mM PMSF and the protease inhibitors tosylphenylalanine chloromethyl ketone, tosyllysine chloromethyl ketone, leupeptin, pepstatin A, antipain and aprotinin, each at 25 µg ml-1] by vigorous shaking with 0·45 mm glass beads in a fast-prep cell breaker (Bio 101; level 5·5 for 25 s). Cell extracts were separated from glass beads and cell debris and collected in an Eppendorf tube by centrifugation, then further clarified by a 13000 g spin for 15 min at 4 °C. The protein concentration of the supernatants was measured at 280 nm and normalized with lysis buffer. Then, 2x SDS-PAGE sample loading buffer was added and samples were boiled for 5 min. Protein samples (50 µg) were fractionated by SDS-PAGE using 8% polyacrylamide gels and transferred to nitrocellulose membranes (Hybond; Amersham). Membranes were probed with anti-Clb2 polyclonal antibodies (kindly provided by D. Kellogg) at 1/2000 dilution in the presence of 5% non-fat milk for 2 h at room temperature. The primary antibody was detected using a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody with the ECL detection system.
Confocal and fluorescence microscopy.
Thin SD-agar medium layers on slides for time-lapse microscopy were prepared as described previously (Jiménez et al., 1998 ). Confocal microscopy was performed with an Olympus IMT-2 microscope attached to a Bio-Rad MRC1000 confocal system. The thickness of confocal sections was 1 µm.
For fluorescence microscopy, cells from exponentially growing cultures were spun, washed once with sterile water and observed. The fluorescence microscope was equipped with a HB-10101AF mercury fluorescent lamp from Nikon. Photographs were taken with a Nikon FX-35A camera and the film used was Ilford 400 ASA. For statistics on cell populations expressing the septin-GFP fusion, a mean of 200 cells were counted for each experiment.
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RESULTS |
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Saccharomyces cerevisiae diploid strains are susceptible to undergoing pseudohyphal growth when starved for nitrogen in solid medium, displaying a growth pattern that diverges from that of budding yeast cells (Kron et al., 1994 ). Yeast growth obeys a bipolar pattern of polarity and is asynchronous, that is mother and daughter cells do not start a new round of budding at the same time due to the fact that cell separation takes place when the daughter has not achieved its maximum size. Cells in pseudohyphae, however, divide synchronously and in a unipolar fashion, probably to facilitate colonization of the medium. A diploid strain on a
1278b background was transformed with pLA10 and examined by time-lapse confocal microscopy for Cdc10-GFP localization in low-nitrogen SLADH agar microlayers. The results of this experiment, shown in Fig. 1(p
s
), revealed that synchronous unipolar growth is also supported by a septin ring that follows a basic dynamic pattern indistinguishable from that observed during the budding of individual yeast cells grown in rich medium.
The START landmark of the cell cycle is essential for septin ring assembly
Reorganizations of cytoskeletal structures that participate in morphogenesis, such as the actin cortical patches, are triggered by cell-cycle-dependent signals (Lew & Reed, 1993 ). The association or dissociation of the yeast main CDK, namely Cdc28, with either G1 (Cln) or mitotic (Clb) cyclins seem to accurately control actin rearrangements throughout the cell cycle (Lew & Reed, 1995
). It has been shown that actin polarization to the pre-bud site depends on the START landmark of the cell cycle, characterized by the appearance of Cln-Cdc28 complexes (Lew & Reed, 1993
). We wished to know whether the appearance of the septin ring at the incipient bud site might also depend on such CDK-dependent signals. If so, cdc28-4 mutant strains, which bear a thermosensitive START-defective specific allele of the yeast CDK, should fail to assemble the septin ring. As shown in Fig. 2
, Cdc10-GFP was correctly localized at the permissive temperature in strain K1414 (more than 90% of the cells showed assembled rings), but could not be detected after the culture was switched to 37 °C for 2 h (more than 90% of the population lacked rings). Statistically, 88% of the cells were budded at 24 °C and, within the unbudded population, 69% bore septin rings, whereas after the switch to 37 °C 92% of the cellular population lacked buds, none of them displaying localized Cdc10-GFP (n=200). This is not due to loss of GFP fluorescence at high temperatures, since the Cdc10-GFP fusion can be readily observed in cells grown at 37 °C (Cid et al., 1998a
; see below). We observed that the morphogenetic effects of the cdc28-4 mutation were dependent on the strain background. In genetic backgrounds different from that of the K1414 strain, derived from crosses of this strain with different wild-type strains, cells occasionally tended to elongate and Cdc10-GFP marks were often detected, although they constituted fairly aberrant structures rather than proper rings (data not shown). Altogether, these data suggest that septin assembly is dependent on a functional G1 cyclin/Cdc28 complex.
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Rho1 is another small GTPase that plays a key role in the regulation of morphogenesis (Drgonová et al., 1996 ; Qadota et al., 1996
; see Cabib et al., 1998
for a review), promoting actin-based polarized secretion and cell wall glucan synthesis. We transformed the strains HNY21 (rho1-104) and OHNY1 (isogenic wild-type control) with the pLA10 plasmid to study septin distribution under the expression of the ts- rho1 mutation. As shown in Fig. 4(a)
and (b)
, after incubation at 37 °C for 2 h, wild-type cells showed the expected proportion of cells displaying single, neck-spanning and separated septin rings. Single rings appeared in unbudded or small-budded cells, whereas in medium- or large-budded cells, the Cdc10-GFP mark extended around the base of both mother and daughter cells as an hourglass-like neck-spanning ring. Separated parallel rings occurred only in large-budded cells (see Fig. 4a
for an illustration of these three types of cells). rho1 mutant cells arrested with a small incipient bud that was encircled by a single Cdc10-GFP ring (Fig. 4a
and b
).
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Septin ring integrity is unaffected by either G2 or M cell cycle arrests
Deregulation of the expression of G1 cyclins affects morphogenesis by altering the dynamics of the actin cytoskeleton (Lew & Reed, 1993 ). At this point we wished to study the effect of the overexpression of Cln2 (a G1 cyclin) in the septin cytoskeleton. With this aim, we constructed strain LAY1, bearing a GAL1-CLN2 cassette integrated in its genome. Incubation of this strain in galactose led to partial cell elongation as a morphogenetic response to a delay in the entry into M phase. This effect was not very dramatic since a single copy of the overexpression cassette would not be expected to cause a serious delay of the cell cycle (Lew & Reed, 1993
). In general, in spite of the morphological alteration, septins were properly located under these conditions (Fig. 5e
). Only a careful statistical study of the cell population revealed that the proportion of cells displaying a single ring was slightly higher in galactose than in glucose medium, in detriment to the population with neck-spanning or separated neck rings (Fig. 5a
). This observation probably reflects a partial accumulation of cells in late G1. A more drastic pre-mitotic arrest can be achieved by activation of the DNA-damage cell cycle checkpoint (see Longhese et al., 1998
for a review). The checkpoint mechanism is activated by failures during DNA replication, which are thought to induce an inactivation of the Cdc28-Clb kinase until the mistake is corrected, thus subordinating mitosis to prior essential events in the cell cycle. We analysed septin-GFP distribution in the YJJ21 strain, which bears a mutation in the cdc13 gene that encodes a telomere-binding protein whose dysfunction causes the activation of the DNA-damage/repair checkpoint (Nugent et al., 1996
). Incubation of the mutant at the non-permissive temperature led to homogeneous arrest of the cells as doublets with a continuous neck-spanning septin ring (Fig. 5b
and f
).
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Septin ring separation at cytokinesis depends on the function of late mitotic regulators
We simultaneously introduced the pLA10H and the pGAL1-CLB2 plasmids in the FY1679-1a strain to study the influence of B cyclin overexpression in Cdc10-GFP dynamics. As depicted in the graph in Fig. 6(a), incubation of the transformants in galactose led to the accumulation of cells with a continuous neck-spanning septin ring, probably as a consequence of the elongation of the M phase. The expression of a non-degradable version of the cyclin, CLB2(db
) (Surana et al., 1993
), under its own promoter led to a similar pattern (data not shown). Cdc15 is a protein kinase essential for the exit from mitosis, arresting cells in late anaphase (Schweitzer & Philippsen, 1991
; Surana et al., 1993
). In a previous study, we reported that cdc15 mutants were unable to septate and to reassemble septins at new sites of polarized growth (Jiménez et al., 1998
). A side observation was that cdc15-arrested mutants, like cdc13- cdc16- cdc23- or nocodazole-arrested cells, were not able to give rise to the double clearly separated septin ring that is characteristic of the cytokinetic stage. The same phenomenon was observed in cdc14-1 mutants (Fig. 6b
) defective in a protein phosphatase that plays an essential role in mitotic exit (Shou et al., 1999
; Visintin et al., 1999
), as determined after transformation of the 15DAU strain with pLA10 and analysis of Cdc10-GFP distribution at 37 °C.
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DISCUSSION |
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Fig. 7 summarizes the hypotheses suggested by the results in this work. Although it is not shown in Fig. 7
to avoid complication, in cells growing exponentially the disappearance of the septin ring remaining from the previous cytokinesis site usually overlaps in time with the assembly of the new ring at an adjacent spot (see Fig. 1f
, in which both old and new rings co-exist at a certain stage). In a previous report (Jiménez et al., 1998
) we showed that in late mitotic mutants (cdc14, cdc15, etc.) cytokinetic-defective cells can rebud but they cannot remove septins from the aborted cytokinesis site. This observation suggests that the disassembly of the septin ring relies on signals left by the cytokinetic machinery. The total lack of assembled septin structures in cdc28-4 mutants indicates that the disassembly of septins at the previous cytokinetic site does not depend on the function of G1 cyclin-Cdc28 kinase. At the same time, this indicates that septin assembly to the selected bud site is dependent on such activity. Our observation that both the loss of function and the expression of constitutively activated Cdc42 alter septin distribution indicates that this small GTPase plays a role in directing the proper assembly of this structure. The facts that Cla4, a protein kinase from the PAK family that interacts with Cdc42, is important for proper bud neck assembly (Cvrcková et al., 1995
) and that certain mutant alleles of Cdc42 display a behaviour similar to that of cla4 mutants (Richman et al., 1999
) suggest that Cla4 may be a mediator in Cdc42 function in the co-ordination of septin ring formation. We also show that the Rho1 GTPase plays its essential role in bud emergence once septins are properly assembled, further supporting the notion that Rho1 functions in a stage downstream of Cdc42, as proposed by Chant (1994)
. As a consequence, the function of Rho1 is not essential for septin assembly. However, the aberrant septin distribution that we observed in mutants in the putative Rho1-GAP bem2 suggests that hyperactive Rho1 may cause discoordination of morphogenetic events throughout cell division, leading to general perturbations in cytoskeletal dynamics. Still, the role of Bem2 in morphogenesis could be something other than its regulation of Rho1, as suggested by the pleiotropic bem2 phenotype. In spite of their occasionally altered thickness, shape or localization, septin rings are assembled at the bud site in bem2 mutants, reinforcing the idea that Rho1-dependent signals play no role in septin assembly.
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We show here that the eventual separation of these rings depends on the onset of the molecular mechanisms that trigger exit from mitosis, namely B cyclin removal. We base this conclusion on the fact that cdc14 and cdc15 mutants, as well as APC (cdc16 and cdc23) mutants, are essential for the splitting of the septin ring into two parallel structures and that this phenomenon occurs simultaneously with Clb2 destruction. The observation that long incubation times at the non-permissive temperature of cdc14, cdc15, cdc16 or cdc23 mutants result in overspread neck-spanning septin structures suggests that the active Cdc28-B cyclin complex participates in septin ring expansion towards both sides of the neck. As suggested by the predominance of the same double-sided rings in either nocodazole- or cdc13-arrested cells, the cell cycle checkpoints that respond to spindle disassembly and DNA damage, respectively, do not influence the development of the septin cytoskeleton at this stage. This is not surprising in nocodazole-arrested cells, since damage of the spindle or its motor structures is known to cause cell cycle arrest in anaphase, apparently at the same morphogenetic stage as mutations in APC components. However, activation of the DNA-damage cell cycle checkpoint by mutations in cdc13 is known to arrest the cell cycle earlier, namely at the G2/M transition (Garvik et al., 1995 ). Although the cell does not undergo nuclear mitosis in cdc13 mutants, bud growth persists and the septin cytoskeleton evolves to the same stage as nocodazole- or even cdc14-arrested cells. One way of understanding this is that the signal that determines isotropic bud growth commits the daughter cell to reach its maximum size regardless of nuclear events. This hypothesis would assume that the formation and maintenance of a symmetric neck-spanning septin structure is a consequence of the endowment of isotropic bud growth. In summary, the co-ordination of morphogenesis throughout the cell cycle might rely on three essential landmarks, as postulated by Lew & Reed (1995)
: bud emergence, promoted by G1 cyclins; the switch from polar to isotropic growth, determined by B cyclins; and cytokinesis, triggered by the degradation of B cyclins. In parallel, we propose that the same sequence of oscillations in cyclin levels would determine, respectively, the assembly of the septin ring, its development into a symmetric structure and finally its separation into two distinct parallel rings.
The role of septins in the control of morphogenesis during cell division
The role of the septin cytoskeleton in defining the pattern of polarity has been postulated on the basis that septin mutants lose cell polarity and that bud site selection markers, such as Bud3, Bud4 and Bud10, colocalize with septins (Flescher et al., 1993 ; Chant et al., 1995
; Sanders & Herskowitz, 1996
; Halme et al., 1996
; Roemer et al., 1996
). Once the bud site is chosen, the combined functions of the actin and septin cytoskeletal structures seem to direct bud emergence and growth. At the time of bud emergence, actin and septin coincide at the budding site, but actin cortical patches move to the growing tip as the bud emerges, while septins remain unaltered at the base of the bud. An interesting view for understanding morphogenesis at this stage would be to consider the assembled septin ring as the border of the morphogenetically active site at the cell surface. Although this view has proven to be applicable to later stages of the cell cycle, during bud development (Barral et al., 2000
; Takizawa et al., 2000
), it fails to ring true at the initial stage, since bud emergence, regardless of polarity, is properly achieved in septin mutants (Hartwell, 1971
). Assuming that the septin cytoskeleton is dispensable for bud emergence, why does Cdc42 recruit septins at the pre-bud site at that particular stage of cell cycle? According to the phenotype of septin mutants, which leads to hyperpolarized bud growth, the septin cytoskeleton is essential for the transition from polar to isotropic bud growth, a process that relies on the activation of Clb-Cdc28 complexes (Lew & Reed, 1993
). In fact, septin-associated components of signal transduction pathways have been recently associated with Swe1-dependent activation of Cdc28 (Barral et al., 1999
; Shulewitz et al., 1999
; McMillan et al., 1999
), defining a morphogenesis-dependent cell cycle checkpoint. A sensible hypothesis is that septins would be laid at the bud site to participate in bud-emergence-dependent signals that activate the checkpoint pathways for dephosphorylation and activation of B cyclin-Cdc28, which in turn promotes isotropic bud growth by means of yet unknown mechanisms.
The other stage of the cell cycle in which septins play a crucial role is cytokinesis. Our previous results suggested that the septation mechanisms are not blocked in septin mutants, but the spatial landmark to localize such mechanisms is lost (Cid et al., 1998a ). Although septin mutants are unable to form septa properly, septins do not seem to be components of the contractile structure assembled at the time of septation, but submembrane marks for its proper localization. Rather than contracting at the cytokinetic stage, the septin hourglass-shaped structure splits into two differentiated rings. It has been demonstrated that an actomyosin contractile ring is located in between both septin rings at that stage (Lippincott & Li, 1998
; Bi et al., 1998
). This observation further suggests that the role of the septin cytoskeleton is to locate the cytokinetic machinery at the proper plane. Further research on septin-interacting components and the combined dynamics of both the septin and actin cytoskeletal scaffolds should shed light on the important issue of cell-cycle-dependent regulation of morphogenesis. In this sense, the yeast model can be expected to reveal important clues for the developmental behaviour of higher eukaryotic cells.
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ACKNOWLEDGEMENTS |
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Received 25 October 2000;
revised 19 February 2001;
accepted 26 February 2001.