Poles Apart: Prokaryotic Polar Organelles and Their Spatial Regulation

While polar organelles hold the key to understanding the fundamentals of cell polarity and cell biological principles in general, they have served in the past merely for taxonomical purposes. Here, we highlight recent efforts in unraveling the molecular basis of polar organelle positioning in bacterial cells. Specifically, we detail the role of members of the Ras-like GTPase superfamily and coiled-coil-rich scaffolding proteins in modulating bacterial cell polarity and in recruiting effector proteins to polar sites. Such roles are well established for eukaryotic cells, but not for bacterial cells that are generally considered diffusion-limited. Studies on spatial regulation of protein positioning in bacterial cells, though still in their infancy, will undoubtedly experience a surge of interest, as comprehensive localization screens have yielded an extensive list of (polarly) localized proteins, potentially reflecting subcellular sites of functional specialization predicted for organelles.Since the first electron micrographs that revealed flagella at the cell poles of bacteria, we have known that bacterial cells are polarized and that they are able to decode the underlying positional information to confine the assembly of an extracellular organelle to a polar cellular site (Fig. 1). Foraging into this unknown territory has been challenging, but recent efforts that exploit the power of bacterial genetics along with modern imaging methods to visualize proteins in the minute bacterial cells has yielded several enticing entry points to dissect polarity-based mechanisms and explore potentially contributing subdiffusive characteristics (Golding and Cox 2006).Open in a separate windowFigure 1.Transmission electron micrograph (taken by Jeff Skerker) of a Caulobacter crescentus swarmer cell showing the polar pili (empty arrowheads), the polar flagellum with the flagellar filament (filled arrowheads), and the hook (white arrow) (see Fig. 2A).While polar organelles are a visual manifestation of polarity, it is important to point out that polarity can also be inherent to cells, at least in molecular terms, even in the absence of discernible polar structures. In other words, molecular anatomy can reveal that a bacterial cell, such as an Escherichia coli cell, features specialized protein complexes at or near the poles, despite a perfectly symmetrical morphology (Maddock and Shapiro 1993; Lindner et al. 2008). Such systemic polarization in bacteria, likely stemming from the distinctive division history of each pole, has the potential to be widespread and to be exploited for positioning of polar organelles and protein complexes. As excellent reviews have been published detailing the interplay between cell polarity and protein localization (Dworkin 2009; Shapiro et al. 2009; Kaiser et al. 2010; Rudner and Losick 2010), here we focus on recent progress in understanding the function and localization of spatial regulators of polar organelles. Considering that the ever-growing list of polar protein complexes emerging from systematic and comprehensive localization studies (Kitagawa et al. 2005; Russell and Keiler 2008; Werner et al. 2009; Hughes et al. 2010) is suggestive of multiple polarly confined (organelle-like) functions, understanding their spatial regulation is also of critical relevance in the realm of medical bacteriology, as many virulence determinants also underlie polarity (Goldberg et al. 1993; Scott et al. 2001; Judd et al. 2005; Jain et al. 2006; Jaumouille et al. 2008; Carlsson et al. 2009). Below, we highlight a few prominent examples of overtly polar organelles and the proteins known to date that regulate their polar positioning.     

Cell Division in Yeasts: Movement of Organelles Associated with Cell Plate Growth of Schizosaccharomyces pombe

Electron microscopy of dividing fission yeast cells shows establishment of an annular rudiment (AR) of electron-transparent material under the old cell wall as the first sign of elaboration of the cell plate. The AR grows centripetally, finally closing at the mid-point of the cell. During the inward growth of the AR it is thickened by addition of denser material which becomes the scar plug after fission; the electron-transparent material is lost at fission. Lying always between the cytoplasmic membrane and the cell wall is a dark layer of variable thickness. This layer becomes markedly thickened into a fillet at the base of the centripetally growing cell plate. The fission process begins after the cell plate is completely elaborated. One striking feature of fission is the migration of dense material from the fillet at the base of the cell plate outwardly through the matrix of the cell wall to its final resting place as a dark ring, a "fuscannel," adjacent to the fission scar. The inclusion of Golgi bodies in many sections suggests their involvement in cell plate elaboration, presumably through production of the dense bodies which are seen to fuse with the dark layer proximal to the growing cell plate.     

Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

Dynamic Interplay of Spectrosome and Centrosome Organelles in Asymmetric Stem Cell Divisions

Stem cells have remarkable self-renewal ability and differentiation potency, which are critical for tissue repair and tissue homeostasis. Recently it has been found, in many systems (e.g. gut, neurons, and hematopoietic stem cells), that the self-renewal and differentiation balance is maintained when the stem cells divide asymmetrically. Drosophila male germline stem cells (GSCs), one of the best characterized model systems with well-defined stem cell niches, were reported to divide asymmetrically, where centrosome plays an important role. Utilizing time-lapse live cell imaging, customized tracking, and image processing programs, we found that most acentrosomal GSCs have the spectrosomes reposition from the basal end (wild type) to the apical end close to hub-GSC interface (acentrosomal GSCs). In addition, these apically positioned spectrosomes were mostly stationary while the basally positioned spectrosomes were mobile. For acentrosomal GSCs, their mitotic spindles were still highly oriented and divided asymmetrically with longer mitosis duration, resulting in asymmetric divisions. Moreover, when the spectrosome was knocked out, the centrosomes velocity decreased and centrosomes located closer to hub-GSC interface. We propose that in male GSCs, the spectrosome recruited to the apical end plays a complimentary role in ensuring proper spindle orientation when centrosome function is compromised.