Localized Cardiolipin Synthesis is Required for the Assembly of MreB During the Polarized Cell Division of Chlamydia trachomatis

Pathogenic Chlamydia species are coccoid bacteria that use the rod-shape determining protein MreB to direct septal peptidoglycan synthesis during their polarized cell division process. How the site of polarized budding is determined in this bacterium, where contextual features like membrane curvature are seemingly identical, is unclear. We hypothesized that the accumulation of the phospholipid, cardiolipin (CL), in specific regions of the cell membrane induces localized membrane changes that trigger the recruitment of MreB to the site where the bud will arise. To test this, we ectopically expressed cardiolipin synthase (Cls) and observed a polar distribution for this enzyme in Chlamydia trachomatis. In early division intermediates, Cls was restricted to the bud site where MreB is localized and peptidoglycan synthesis is initiated. The localization profile of Cls throughout division mimicked the distribution of lipids that stain with NAO, a dye that labels CL. Treatment of Chlamydia with 3-,6-dinonylneamine (diNN), an antibiotic targeting CL-containing membrane domains, resulted in redistribution of Cls and NAO-staining phospholipids. In addition, MreB localization was altered by diNN treatment, suggesting an upstream regulatory role for CL-containing membranes in directing the assembly of MreB. This hypothesis is consistent with the observation that the clustered localization of Cls is not dependent upon MreB function or peptidoglycan synthesis. Furthermore, expression of a CL-binding protein at the inner membrane of C. trachomatis dramatically inhibited bacterial growth supporting the importance of CL in the division process. Our findings implicate a critical role for localized CL synthesis in driving MreB assembly at the bud site during the polarized cell division of Chlamydia.

Septin Assembly and Remodeling at the Cell Division Site During the Cell Cycle

The septin family of proteins can assemble into filaments that further organize into different higher order structures to perform a variety of different functions in different cell types and organisms. In the budding yeast Saccharomyces cerevisiae, the septins localize to the presumptive bud site as a cortical ring prior to bud emergence, expand into an hourglass at the bud neck (cell division site) during bud growth, and finally “split” into a double ring sandwiching the cell division machinery during cytokinesis. While much work has been done to understand the functions and molecular makeups of these structures, the mechanisms underlying the transitions from one structure to another have largely remained elusive. Recent studies involving advanced imaging and in vitro reconstitution have begun to reveal the vast complexity involved in the regulation of these structural transitions, which defines the focus of discussion in this mini-review.

Haspin Modulates the G2/M Transition Delay in Response to Polarization Failures in Budding Yeast

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.

Overexpression of the Aspergillus fumigatus Small GTPase, RsrA, Promotes Polarity Establishment during Germination

Cell polarization comprises highly controlled processes and occurs in most eukaryotic organisms. In yeast, the processes of budding, mating and filamentation require coordinated mechanisms leading to polarized growth. Filamentous fungi, such as Aspergillus fumigatus, are an extreme example of cell polarization, essential for both vegetative and pathogenic growth. A major regulator of polarized growth in yeast is the small GTPase Rsr1, which is essential for bud-site selection. Here, we show that deletion of the putative A. fumigatus ortholog, rsrA, causes only a modest reduction of growth rate and delay in germ tube emergence. In contrast, overexpression of rsrA results in a morphogenesis defect, characterized by a significant delay in polarity establishment followed by the establishment of multiple growth axes. This aberrant phenotype is reversed when rsrA expression levels are decreased, suggesting that correct regulation of RsrA activity is crucial for accurate patterning of polarity establishment. Despite this finding, deletion or overexpression of rsrA resulted in no changes of A. fumigatus virulence attributes in a mouse model of invasive aspergillosis. Additional mutational analyses revealed that RsrA cooperates genetically with the small GTPase, RasA, to support A. fumigatus viability.

Saccharomyces spores are born prepolarized to outgrow away from spore-spore connections and penetrate the ascus wall

1.AbstractHow non-spore haploid Saccharomyces cells choose sites of budding and polarize towards pheromone signals in order to mate has been a subject of intense study. Unlike non-spore haploids, sibling spores produced via meiosis and sporulation by a diploid cell are physically interconnected and encased in a sac derived from the old cell wall of the diploid, called the ascus. Non-spore haploids bud adjacent to previous sites of budding, relying on stable cortical landmarks laid down during prior divisions, but since spore membranes are made de novo it was assumed that, as is known for fission yeast, Saccharomyces spores break symmetry and polarize at random locations. Here we show that this assumption is incorrect: Saccharomyces cerevisiae spores are born prepolarized to outgrow, prior to budding or mating, away from interspore bridges. Consequently, when spores bud within an intact ascus, their buds locally penetrate the ascus wall, and when they mate, the resulting zygotes adopt a unique morphology reflective of re-polarization towards pheromone, which we dub the derrière. Long-lived cortical foci containing the septin Cdc10 mark polarity sites, but the canonical bud site selection program is dispensable for spore polarity, thus the origin and molecular composition of these landmarks remain unknown. These findings demand further investigation of previously overlooked mechanisms of polarity establishment and local cell wall digestion, and highlight how a key step in the Saccharomyces life cycle has been historically neglected.

Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1

The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.

Cdc24, the source of active Cdc42, transiently interacts with septins to create a positive feedback between septin assembly and bud site formation in yeast cells

Yeast cells select at the beginning of each cell cycle the position of their new bud. The recruitment of the septins to this prospective bud site (PBS) is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. The septin-rods follow hereby the high concentration of Cdc42GTP that is generated by the focused location of its GEF Cdc24. We show that Cdc24 not only activates Cdc42 but temporarily interacts shortly before budding with Cdc11, the subunit that caps septin rods at its both ends. Mutations in Cdc24 that reduce the affinity to Cdc11 impair septin assembly and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize into the ring, Cdc24 transfers to its center and directs from there the further outgrowth of the membrane.

Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1

Establishing cell polarity is critical for growth and development of most organisms. The Cdc42 GTPase plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site (i.e. bud site) is selected in the G1 phase, which determines the axis of cell polarization. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet the mechanism underlying spatial cue-directed cell polarization is not fully understood. Here, we show that Rsr1 associates with Bem1, a scaffold protein, preferentially in its GDP-bound state in early G1. This interaction involves a part of the Bem1 Phox homology (PX) domain, which overlaps with a region previously shown to interact with Exo70, an exocyst component. Furthermore, overexpression of the constitutively GDP-bound Rsr1 interferes with Bem1’s association with Exo70 and inhibits Bem1-dependent Exo70 polarization. We propose that Rsr1 plays a delicate role in coordination of spatial and temporal regulation of polarity establishment via its GTP- and GDP-bound states.