Septins: molecular partitioning and the generation of cellular asymmetry
© McMurray and Thorner; licensee BioMed Central Ltd. 2009
Received: 9 August 2009
Accepted: 26 August 2009
Published: 26 August 2009
During division, certain cellular contents can be distributed unequally; daughter cells with different fates have different needs. Septins are proteins that participate in the establishment and maintenance of asymmetry during cell morphogenesis, thereby contributing to the unequal partitioning of cellular contents during division. The septins themselves provide a paradigm for studying how elaborate multi-component structures are assembled, dynamically modified, and segregated through each cell division cycle and during development. Here we review our current understanding of the supramolecular organization of septins, the function of septins in cellular compartmentalization, and the mechanisms that control assembly, dynamics, and inheritance of higher-order septin structures, with particular emphasis on recent findings made in budding yeast (Saccharomyces cerevisiae).
Overview: Jumping Through Hoops
After anaphase, cytokinesis completes the process of producing two cells from one. For proliferation to occur, each daughter cell must receive at every such mitosis all of the requisite components essential for subsequent division. During development, by contrast, certain daughter cells inherit particular cellular constituents differentially, which can influence their fate. Within non-dividing cells, establishment of cellular asymmetry ("polarity") requires spatial segregation of molecular components, and this selective partitioning may be a fundamental feature of life . Despite its universal importance, many aspects of how such subcellular asymmetry is generated remain poorly understood at the mechanistic level. In a number of biological contexts, a set of conserved proteins, called septins, has emerged as a central player in polarity determination and asymmetric cell division.
The septins are a family of GTP-binding proteins found in nearly all eukaryotes (higher plants are the main exception) . A given septin assembles with other septins into a linear hetero-oligomeric complex ("rod"), and rods can associate end-to-end to form longer polymers ("filaments") . For example, the S. cerevisiae rod capable of polymerization in vitro is a hetero-octamer composed of four different gene products in the following order: Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 . Targeted localization directs assembly of septin ensembles at particular sites, and septin-containing structures have been implicated in a wide variety of cellular processes . Septin-based structures seem to perform, in essence, two non-catalytic roles. First, septin structures serve as scaffolds for the recruitment of non-septin factors, i.e., they participate in cell morphogenesis and cell division via their direct physical interaction with various enzymes and regulatory proteins. Second, septin structures that are closely associated with membranes can serve as barriers that restrict the movement of certain integral membrane proteins, i.e., localization of such membrane proteins is septin-dependent, but does not seem to involve their stable binding to the septins [6–8].
Mechanistically, how septins create a barrier to diffusion along a membrane remains largely unknown. However, the ability of septin complexes to polymerize into filaments provides one reasonable possibility. For example, at the EM level, the septin collar at the bud neck appears to comprise a highly ordered array of continuous circumferential filaments ("hoops")  and these are present at the stage of the yeast cell cycle when diffusion of cortical components between a mother and its bud is demonstrably restricted . Yeast septin rods (either isolated from S. cerevisiae  or prepared by expression in and purification from E. coli [4, 21–23]) are able to self-assemble under the right conditions (salt concentration ≤ 150 mM) into filaments that strikingly resemble the neck filaments, suggesting that these septin filaments are themselves the primary constituents of the collar hoops. Indeed, indirect immunofluorescence using anti-septin antibodies  and examination of cells expression GFP-tagged septins  confirm that the collar contains the vast majority of the septins present in a budded cell. Moreover, the collar filaments are closely apposed to the PM . It has been suggested that membrane association of septins is mediated via their interaction with phosphatidylinositol-4,5-bis phosphate (PtdIns4,5P2) in both mammalian cells  and yeast . Consistent with this property of the septins and their location in a budded cell, there is evidence that PtdIns4,5P2 is enriched in the PM at the bud neck . Furthermore, forced wholesale conversion of the PM PtdIns4,5P2 pool to PtdIns3,4,5P3 causes the detachment of septins from the bud neck and the formation of coils and rings in the cytosol . Preparations of human septins are purportedly capable of remodeling large synthetic membrane vesicles into tubular projections by wrapping around the tubes in a PtdIns4,5P2-dependent manner ; however, the possibility of contamination by other proteins (like ESCRT III components) capable of membrane tubulation has not been scrupulously eliminated. In any event, the clear-cut implication of these collective findings is that, by associating tightly with the PM, continuous septin filaments in the collar at the bud neck could act like the fences in a corral to physically constrain the movement of both lipids and proteins, thereby preventing their free passage between a mother cell and its bud [31, 32] (Figure 1). The septin filaments in the collar appear to coat the PM at the neck, but project less than 10–20 nm into the cytosol; at least one septin-associated peripheral membrane protein, Bud6, is required to impose the ER and NE barriers, but not the PM barrier [11, 12], indicating that different factors are involved in establishing the septin-dependent diffusion barriers at the PM and at other membranes.
A second concern raised about the corral model has to do with a dispute about the orientation of the bud neck filaments relative to the mother-bud axis. For a corral to prevent movement of factors from one side of the isthmus to the other, at least some of the filaments in the collar should run perpendicular to that axis, i.e., circumferential to the neck (Figure 1), consistent with the interpretation of the original EM images in which the filaments were first detected . However, it has been posited, instead, that the filaments run parallel to the mother-bud axis, and that the filaments only appear circumferential because, according to this view, the filaments are in perfect side-by-side register and their constituent septin subunits have differential avidity for the stain used for EM visualization [34, 35]. To attempt to bolster this argument, the orientation in vivo of septin filaments containing GFP-tagged Cdc12 or Cdc3 was examined by measurement of the fluorescence polarization of this fluorophore, compared to a "standard" (bundled filaments of the same proteins prepared by purification and assembly in vitro) . By this criterion, the filaments in the collar appeared to be oriented parallel to the mother-bud axis. Interestingly, in these studies, as judged by a 90° shift in the fluorescence anisotropy of the GFP-tagged septins in vivo at the onset of cytokinesis, the orientation of the septin filaments seemed to undergo a 90° rotation when the collar is split into two rings , consistent with the rings now comprising circumferential filaments (as would be needed for a septin-based corral). Yet, the barrier function of the septin collar is also exerted prior to cytokinesis; so, the question remained of how filaments parallel to the mother-bud axis can do so. In the fluorescence polarization studies, it was assumed that the orientation of the GFP relative to the septin to which it is attached remains fixed and that the chimera behaves as one rigid object. However, more recent ultrastructural analysis has demonstrated that the carboxyl-terminal portion of the septin proteins (to which the GFP was attached in the polarized fluorescence experiments) is flexible and able to rotate freely relative to the filament axis [4, 22, 37]. Thus, the most parsimonious conclusion that reconciles the available EM and fluorescence polarization data is to view the septin collar as an array of circumferential filaments, as originally proposed, which is resolved at the onset of cytokinesis into a pair of rings that are also composed of circumferential filaments, but in which the flexible septin carboxy termini have undergone a 90° rotation (Figures 1 and 2). Accordingly, at both stages of the cell cycle, the collar and the rings have the same underlying mechanistic basis for exerting a diffusion barrier function. The shift in orientation of the septin tails may simply reflect a conformational change induced by their association with different sets of proteins and/or lipids, or different types or extents of cell cycle stage-specific post-translational modifications.
A third objection to the idea that an essential function of the septins is the formation of circumferential filaments that establish a diffusion barrier was a study that concluded that assembly of septin filaments per se was not essential for S. cerevisiae cell division . The data in this study that seemed the most persuasive at the time was the proliferative ability of cells that lack a particular septin (either Cdc10 or Cdc11) and in which no prominent array of neck filaments was visible by EM, and from which purified septin complexes were unable to polymerize into filaments in vitro [20, 23, 27, 38–40]. Considering that we now appreciate that, in yeast, the building block of filaments is a linear (single subunit-wide) hetero-octameric rod that polymerizes end-on-end [4, 22], deletion of any single subunit might be expected to preclude polymerization. However, when expressed in and purified from bacteria, complexes of yeast septins lacking either Cdc10 or Cdc11 have been reported to form filaments in vitro, although the protein concentrations required are higher and the resulting filaments are less organized than observed for the complete four-subunit complex [21, 23]. Moreover, although the majority of cdc10 Δ cells in the study of Frazier et al. did not exhibit a pronounced array of neck filaments visible by EM, one cell did display a repeated pattern of cortical profiles reminiscent of neck filaments . Furthermore, in most cdc10 Δ cells, the remaining septins form an apparently continuous collar at the bud neck [20, 23]. Hence, there is evidence to suggest that septin filament assembly in vivo is more forgiving to the absence of constituent subunits than previously thought and, in such cases, detection of neck filaments may require less harsh fixation methods and/or more sensitive techniques. Thus, the findings initially reported by Frazier et al.  do not compellingly exclude the possibility that septin filaments form and are essential for proliferation, and that an essential function of these filaments is the creation of a diffusion barrier.
A fourth finding that has been raised as an argument against the necessity for formation of circumferential neck filaments was the fact that in cells lacking a particular bud neck-associated protein kinase, Gin4, the septins appear at the neck as a series of roughly evenly spaced bars running along the mother-bud axis (a "collar of bars") , instead of the uniform, hourglass-shaped distribution of septins seen in wild-type cells. This arrangement clearly seems incompatible with a corral-type mother-bud diffusion barrier, yet cells containing such abnormal septin-based structures are able to proliferate. The "collar of bars" phenotype is only displayed by a small fraction of the population of gin4 Δ cells, but is more penetrant when gin4 Δ cells are grown at high temperatures. At any given time, more cells display this aberrant septin assembly than display cytokinesis defects, which was taken as evidence that such a collar of septin bars was sufficient for septin function in cell division . On this basis, it was concluded that a hoop-like arrangement of the filaments in the collar is not essential for cell division in budding yeast. The major flaws in this logic are the assumptions that, for every cell found to have a collar of bars, this aberrant structure is the best that the cell will ever assemble during that particular attempt at cell division, and that such cells actually then divide. In fact, however, time-lapse microscopy of gin4 Δ cells expressing a GFP-tagged septin reveals that, in the cells where such bars form, division is delayed, followed either by resolution into a uniform collar and resumption of cytokinesis, or a terminal arrest if the aberrant bar structures persist (our unpublished observations). Thus, it appears that only a uniform septin assembly consistent with circumferential neck filaments is capable of supporting proper cell division.
Versatile Frameworks for Cellular Compartmentation
Cellular subdivision is not an event restricted to the act of cytokinesis during mitotic proliferation. In yeast, for example, meiotic nuclear division is accompanied by a cellularization process, dubbed sporulation . Each one of the four haploid nuclei resulting from meiosis becomes surrounded by a new plasma membrane and cell wall, and these envelopes also encase other cellular components necessary for spore viability and germination. During yeast sporulation, septins are found in a series of structures that assemble at the leading edge of the developing spore membrane [39, 42, 43], where they appear strategically positioned to direct proper localization of the enzymes and regulatory factors directly responsible for spore membrane and wall deposition. PtdIns4,5P2 is highly enriched in these pre-spore membranes , suggesting some common features of the mechanism by which septins interact with those membranes that undergo remodeling during mitotic and meiotic division. However, how this localized recruitment is achieved and why the septins concentrate only in membrane regions undergoing active reorganization is not well understood. Septins appear to interact intimately with microtubules in sporulating cells  and phosphoprotein phosphatase 1 (PP1)-dependent dephosphorylation of as yet unidentified substrates is also critical for proper septin organization during sporulation [41, 45, 46]. Indeed, septin dynamics during spore formation cannot all be explained by the distribution of PtdIns4,5P2 because this lipid is also present in other parts of the developing spore membrane during sporulation  and, likewise, is present in other regions of the plasma membrane during mitotic division. It is possible that the converse model may apply. If septins restrict the diffusion of membrane phospholipids, then the observed PtdIns4,5P2 enrichment at the prospore membrane may be imposed by the concentration of septins there, and not vice versa. However, it is not known if septin-based structures impose a diffusion barrier at the prospore membrane, or whether the sporulation-specific septin complexes, which contain two additional meiosis-specific septins, Spr3 and Spr28 [39, 47], polymerize into filaments.
Partitioning of a Complex Protein Assembly during Cell Division: Septins as a Paradigm
As described above, septin-based structures play important roles in cytokinesis, cell compartmentalization, and cell polarity. At the same time, these elaborate multi-protein ensembles provide an opportunity to understand how complex supramolecular structures are segregated during cell division . During each division of a yeast cell, the five mitotically-expressed septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) co-assemble into a ring that marks the bud site. Concurrently with bud growth, the ring expands into the hourglass-shaped collar that lines the isthmus between the mother cell and its daughter. At cytokinesis, the collar transforms into two rings that demarcate each side of the bud neck (Figures 1 and 2). As mentioned in the preceding section, two additional septin genes are turned on during sporulation, and their products (Spr3 and Spr28) co-assemble with some of the mitotically-expressed subunits (and exclude others) , forming a series of structures that ultimately disappear when, upon germination, a spore resumes mitotic division . Below, we consider what is currently known about the mechanisms by which the septin proteins and the structures of which they are composed are inherited through mitotic and meiotic cell divisions.
Septin Modifications Accompany and Direct Higher-Order Organizational Transitions
Certain cellular factors cannot persist and be passively segregated into daughter cells because their presence would be incompatible with orderly cell division or with the onset of a developmental transition. Cyclins are a good example. These proteins drive the events of mitosis and cytokinesis by directing cyclin-dependent kinases (Cdks) to the substrates whose phosphorylation is rate-limiting for these events. Hence, execution of the cell cycle requires that cyclins be destroyed in the proper temporal and spatial order, thereby yielding new-born daughters competent to undergo terminal differentiation or to initiate their own first division (by commencing reiteration of the same program of cyclin expression).
All five septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) in budding yeast persist throughout the cell division cycle and co-localize indistinguishably at every cell cycle stage . Therefore, unlike cyclins, the complement of septins does not undergo any dramatic change during passage through each cell cycle transition. However, the septins do undergo multiple cell cycle stage-specific modifications that coincide with the dramatic reorganizations of septin-based structures that occur concurrently with progression through the cell division cycle (Figure 2). Thus, it seems reasonable to propose that these modifications affect intermolecular interactions among the septins themselves and/or association of septins with other cellular factors, thereby systematically altering the architecture and components present in septin-based structures at different stages of the cell cycle.
Protein-modifying enzymes and their targets at the Saccharomyces cerevisiae bud neck
Closest mammalian ortholog
NDR family; cell wall morphogenesis, cell separation
Polo/PLK family; cell cycle regulation, cytokinesis, mitotic exit
STE20-like family; activated by GTP-bound Tem1, late nuclear division, mitotic exit
Catalytic subunit of Cdk1; cell cycle progression
B-type cyclin; regulatory subunit for Cdc28; cell cycle progression
NDR family; anaphase-telophase, mitotic exit
Upstream activator of Snf1 and other AMPK-like protein kinases involved in septin assembly
Nim1-related and AMPK-like family; septin collar assembly
Casein kinase I family; located throughout the cell, but concentrated at bud tip and bud neck
Nim1-related and AMPK-like family; morphogenesis checkpoint, Swe1 degradation
Trott AE, Gullbrand B & Thorner J, unpublished results
Nim1-related and AMPK-like family; septin collar assembly
AMPK-like family; negatively regulates mitotic exit in response to a misaligned spindle
Regulatory and substrate targeting subunit of Dbf2 (and Dbf20)
Regulatory and substrate targeting subunit of Cbk1
AB-type cyclins; regulatory subunits for Pho85; cell morphogenesis
Catalytic subunit; transcription, cell morphogenesis
Cell wall integrity; activated by GTP-bound Rho1, localizes to bud neck late in the cell cycle before cell separation
DNA damage checkpoint
Casein kinase I family; a peripheral plasma membrane protein anchored by C-terminal palmitoylation
Regulatory and substrate targeting subunit of phosphoprotein phosphatase (PP) 1; septin reorganization during mating projection formation
B-type regulatory and substrate targeting subunit of PP2A
Catalytic subunit of PP1
Catalytic subunit isoforms of PP2A
B'-type regulatory and substrate targeting subunit of PP2A
A-type regulatory (scaffold) subunit of PP2A
F-box protein for substrate recognition by an SCF (Skp1—Cdc53—Cdc34)-type ubiquitin-protein ligase (E3), targets landmark proteins for destruction after septin ring site is established
PRMT5; type II protein-arginine methyltransferase that generates symmetric N, N'-dimethylarginine residues in its substrates
RING-like domain-containing SUMO-protein ligases (E3); responsible for mother side-specific attachment of SUMO to septins in the collar at the neck at the onset of anaphase
U box domain-containing SUMO-protein ligase (E3); capable of attaching SUMO to septins when Siz1 & Siz2 absent
Small ubiquitin-like modifier; may contribute to regulating disassembly of septin collar and/or association of other proteins with septins in the collar
One way to remove structure- and stage-specific subunit modifications would be to actively reverse them via the action of enzymes that catalyze removal of the modifications. Alternatively, like cyclins, modified septins could simply be destroyed and resynthesized de novo at the appropriate time. Current evidence demonstrates that, in the case of septins, an orderly program of reversible modification (rather than periodic synthesis and degradation) drives the observed changes in organizational state. In mitotically dividing yeast cells, septin polypeptides exhibit a very long half-life [42, 52] and are re-incorporated into every septin-containing structure through multiple successive cell divisions . Furthermore, as mitosis ends, a targeting subunit for phosphoprotein phosphatase 2A (PP2A), Rts1, localizes this enzyme to the split septin rings, promoting dephosphorylation of Shs1 . Consistent with a role for Shs1 dephosphorylation in regulating septin organization at this stage of the cell cycle, when cells lacking Rts1 are propagated at the stressful temperature of 37°C, split rings are misshapen, fail to disassemble properly when a new bud emerges and, more often then not, cytokinesis is not successfully completed . Interestingly, under certain experimental conditions, a single yeast cell can possess multiple buds, and the septin structure that is present at each neck is appropriate to the extent to which that bud has matured . This observation provides suggestive evidence that septin assembly and dynamics are largely influenced by modifications exerted locally rather than responding solely to signals imposed globally across the cell. This same situation is certainly the case in filamentous fungi, like Ashbya gossypii, in which it has been shown that, despite little spatial or temporal separation between them, numerous distinctly different septin-based structures can co-exist in a shared cytoplasm and are subject to regulation by distinct kinases (including the Gin4 ortholog) . Thus, reversible modifications drive transitions in higher-order septin structure, and an inappropriate state of modification (rather than persistence of any septin per se) is deleterious to proper coupling of morphogenesis to cell cycle progression.
Ageism: Septins Do Not Discriminate
As described above, yeast septins are long-lived and re-used in multiple successive divisions. Thus, in each cell, molecules synthesized de novo ("new/naive" septins) co-exist with a substantial population of pre-existing molecules ("old/experienced" septins) that have undergone at least one round of cell cycle-dependent modifications. This situation raises the possibility that old and new septins might be differentially marked, and/or spatially segregated within cellular structures, and thus unequally distributed between a mother cell and its daughter during cell division.
It is known that asymmetric segregation of certain components within other complex macromolecular assemblies can have important consequences. For example, the budding yeast centrosome equivalent, called the spindle pole body (SPB), duplicates in a conservative manner, producing an "old" and a "new" SPB . The old SPB is always the one that is directed bud-ward because cytoplasmic microtubules within the mother cortex direct a regulator of spindle function (Bfa1-Bub2 GAP) specifically to the old SPB . A similar mechanism regulates differential use of the SPBs in the four haploid nuclei produced during meiosis of a diploid yeast cell. As in mitosis, the first two meiotic SPBs differ slightly in age, and both are older than the SPBs generated in the second meiotic division. The temporal order in which the four SPBs are generated dictates the opportunity they have to associate with a packaging factor (Nud1/centriolin), thereby influencing the probability of when they will be encapsulated into spores . Thus, in biology, molecular history can influence subsequent physiological function.
Interestingly, another modification that occurs on Lys residues, and could be mutually exclusive with SUMOylation, is N-acetylation. In this regard, it is noteworthy that the protein-Lys N-acetyltransferase Eco1/Ctf7 is one component identified by mass spectrometry in protein complexes co-purifying with the septin-associated protein kinase Gin4 . Similarly, it has been reported that absence of an otherwise non-essential subunit of the NuA4 histone N-acetyltransferase is synthetically lethal in cells lacking another septin-associated protein kinase, Cla4 . Finally, at least in mammals, the initiator Met is removed from certain septins by the action of methionine exopeptidase and the resulting exposed α-amino group is N-acetylated [65, 66]; a predictive algorithm suggests that all five mitotic S. cerevisiae septins may undergo the same modification .
Certain of the neck-associated protein kinases known to modify septins are restricted to the one side of the neck or the other, suggesting that particular phosphorylation events may also show such a separation. For example, Gin4 (1142 residues) starts off on the mother side of the collar  (Figures 2 and 3), whereas the closely-related (74% identity) enzyme Kcc4 (1032 residues) is found exclusively on the bud side of the collar  (Figure 3). Establishment of this strikingly asymmetric localization occurs early, during assembly of the ring of septins that marks the incipient bud site. For example, septin-binding protein Bni4 associates with the exterior of the ring, whereas Kcc4 is located only at the interior of the ring . Theoretically, if old and new septins were differentially marked, unequal deposition of old and new septins on the outer and inner aspects of the ring (or further differential modifications at the outer and inner edges of the ring) could contribute to establishment of the distinctions between these two zones. In any event, mother-bud asymmetry in septin structures does not appear to be based on any polarity in the organization or arrangement of the constituent septins themselves, but seems instead to be a function of their modification state and/or the nature of their interaction partners.
To address whether differential use of old and new septin molecules might contribute to generating the observed asymmetries, a pulse-chase approach that permits the attachment of fluorescent labels, at will, to existing pools of septin-SNAP-Tag™ fusion proteins was used to distinguish newly synthesized from pre-existing molecules . In the septin structures formed in mitotically dividing cells, new and old septins were found to be intermixed rather homogenously, at least at the resolution of light microscopy . Additionally, old septins were equipartitioned between mother and daughter at each division . Thus, unlike other cellular components, older septins do not accumulate in aging mother cells, even though, ironically enough, trapping other aged, worn-out and damaged cellular components in the mother cell is dependent on the diffusion barrier imposed by the septin collar at the bud neck . The conclusions reached by using time-dependent labeling of SNAP-tagged septins, namely that old septin proteins are reused and recycled many times and and co-localize with newly-made septins, was corroborated using an independent approach for producing and distinguishing between old and new septin based on differential expression of GFP- and mCherry-tagged septins .
The observed intermixing is also consistent with analyses of septin structures performed using fluorescence recovery after photobleaching (FRAP), which indicated extensive mobility of subunits within septin structures at various stages of the cell cycle [53, 69]. Importantly, however, the FRAP method cannot distinguish whether the mobile entity is an individual fluorophore-tagged septin or a larger multimeric complex that contains it. In vitro, purified Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 octameric rods are quite stable and resist dissociation even in buffers of high ionic strength (e.g., 1 M KCl) [4, 20–22], in agreement with the cumulative evidence that such rods are the fundamental building block of septin filaments and higher-order structures seen in vivo. Nonetheless, to examine at molecular resolution whether such rods are stable in vivo once formed, or whether new subunits can be exchanged for old in pre-formed rods, cells expressing a SNAP-tagged septin were pulse-labeled to completion with a biotin affinity label, allowed to assemble into rods, and then allowed to mature through several yeast cell cycles, during which time new (unlabeled) SNAP-tagged molecules are synthesized. The cells were then lysed in high salt and streptavidin capture was used to recover the rods that contain the old (biotin-labeled) septin-SNAP tag subunits. It was found that the majority of these rods also contained newly-made SNAP-tagged subunits, as judged by the fact that they could be labeled subsequently in vitro by incubation with a reactive dye directed against the unoccupied SNAP-tags in those new molecules . Thus, this observation suggests that, in the cell, the subunits within preformed rods undergo dynamic exchange (Figure 2).
Septin Inheritance During Meiotic Divisions
As already recounted above (Figure 2), the transitions of the yeast mitotic division cycle are accompanied by a series of discrete septin-based structures. However, the yeast life cycle includes other development options that also involve formation of unique septin-containing structures distinct from those in mitotic cells. Haploid cells of opposite mating type pair and fuse to form diploids, which can undergo meiosis and sporulation to generate haploid spores. In haploids responding to mating pheromone, normal budding and collar formation are abrograted and septins are found instead at the base and flanks of the polarized structure (mating projection) that forms in such cells. As already mentioned earlier, septin-based structures are formed at the leading edge of the developing spore membranes. Certain septins are essential for all of these events in the yeast life cycle, raising the question of whether a septin subunit made during mitotic division can be recycled for use toward a different developmental purpose, or whether those pre-made proteins are discarded and only newly-made ones employed for such developmental transitions. When the behavior of fluorescent septins, generated by pulse-labeling of septin-SNAP tag fusions, was monitored throughout the course of sporulation, three distinct fates were revealed, depending on the subunit . One subunit (Cdc10) was reused and recycled – that is, molecules synthesized during mitotic proliferation were reincorporated alongside new molecules made during sporulation to build structures near the developing spore membranes. In contrast, a second subunit (Cdc12) made prior to the induction of meiosis also persisted during sporulation, but was not incorporated into the septin-containing structures around the developing spores. Instead, these old Cdc12 molecules were relegated to the ascal cytoplasm and not encapsulated into spores; thus, upon spore germination, the Cdc12 molecules that populate the septins structures needed to support mitotic proliferation were generated only by de novo synthesis. Finally, a third subunit (Spr3) was expressed only during sporulation and replaced the mitosis-specific subunit Cdc12 within the septin complexes in meiotic cells; upon spore germination, robust synthesis of Cdc12, and lack of any further production of Spr3, results in its replacement by Cdc12, thereby excluding Spr3 from mitotic structures. Conversely, robust production of Spr3 in meiotic cells, combined with diminished Cdc12 expression, may contribute, perhaps along with as yet unknown modifications or factors, to excluding Cdc12 from the septin structures on prospore membranes, even in the absence of its proteolytic destruction. In any event, these studies show that dynamic exchange of subunits into and out of septin complexes also occurs during developmental transitions, as well as during the mitotic cell division cycle.
Septins and Histones: Common Principles of Assembly and Inheritance?
It is worth considering the mechanisms of septin assembly and inheritance in light of what is also known about other repeating multi-subunit structures conserved in eukaryotic cells. One such example is the nucleosome, which comprises ~165 base-pairs of double-stranded DNA wrapped around a spherical oligomer of histones, and is the fundamental building block of chromatin and higher-order chromosome structure. Just as the septin hetero-octamer in yeast comprises two copies of each of four different classes of subunits, the nucleosome core is composed of two copies of each of four different classes of histone. Just as the residues in septins can be heavily modified post-translationally, the histones are especially heavily decorated by a variety of post-translational modifications that regulate, among other things, nucleosome accessibilty, higher-order chromatin structure, and coordination of chromosome organization with progression through the cell cycle. Like the septins examined to date, the histones are extremely long-lived in most cellular circumstances [70, 71], demanding that the relevant covalent modifications be reversible. Indeed, existing histone modifications can be enzymatically removed (or counteracted by additional modifications) without disrupting the nucleosome core itself [72, 73], providing non-destructive ways to alter chromatin structure. As observed for septin complexes, exchange of subunits within a nucleosome would allow for replacement of particular subunits with copies carrying a different array of modifications, or with histone variants encoded by distinct genes . The latter echoes the substitution of sporulation-specific septins for mitosis-specific subunits observed in yeast. As is the case with septins, mitotic nucleosome inheritance is symmetrical in the general sense, i.e., the daughters receive an equal share of both the pre-existing and the newly-made histones [72, 73]. However, at higher resolution, the degree to which nucleosomal duplication during S phase is conservative or dispersive remains controversial. Specifically, it has not been definitively established whether the 2:2 tetrameric H3:H4 subcomplex always remains intact or can, in certain situations, split into H3:H4 dimers . A similar uncertainty surrounds septin hetero-octamer dynamics. Are there subunit pairs or sub-complexes that remain associated throughout the lifetime of the constituent proteins (see Figure 2)? Future studies, especially those exploiting recent advances in covalent protein labeling technology, are needed to resolve these issues.
In budding yeast, septin-based structures impose restrictions on the localization of a large number of cellular factors, thereby influencing their distribution and fate during cell division. This influence extends to factors with which the septins do not physically interact and, thus, septin filaments serve not only as scaffolds, but as diffusion barriers. Collectively, by these attributes, septin structures serve as potent cortical organizers. The supramolecular architecture of septin-containing structures themselves undergoes highly regulated transitions coordinated with the yeast cell division cycle and other stages of the life cycle of this organism. During developmental transitions, pre-existing molecules of some subunits inherited from prior cell states are recycled and incorporated into complexes that also contain newly synthesized molecules of the same subunit, whereas incorporation of certain other subunits is restricted to a particular stage and can be irreversibly blocked during developmental transitions. It appears that mechanisms uncovered for regulating septin assembly, dynamics, function and inheritance display principles germane to the behavior of other cellular structures composed of multi-component complexes capable of self-association into polymers.
GTPase activating protein
growth phase 1
growth phase 2
This work was supported by K99 grant GM86603 (to MAM) and by R01 grant GM21841 (to JT) from the National Institutes of Health.
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