Cell Poles Research Articles

Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure.

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, and is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC molecules changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data support a model in which transport of environmental DNA occurs through the entire cell surface by a dynamic pilus, mediating efficient uptake through the cell wall into the periplasm, where DNA diffuses to a cell pole containing the localized transport machinery mediating passage into the cytosol.

Cellular localization of the hybrid pyruvate/2-oxoglutarate dehydrogenase complex in the actinobacterium Corynebacterium glutamicum.

For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.

Bacterial cell-size changes resulting from altering the relative expression of Min proteins

The timing of cell division, and thus cell size in bacteria, is determined in part by the accumulation dynamics of the protein FtsZ, which forms the septal ring. FtsZ localization depends on membrane-associated Min proteins, which inhibit FtsZ binding to the cell pole membrane. Changes in the relative concentrations of Min proteins can disrupt FtsZ binding to the membrane, which in turn can delay cell division until a certain cell size is reached, in which the dynamics of Min proteins frees the cell membrane long enough to allow FtsZ ring formation. Here, we study the effect of Min proteins relative expression on the dynamics of FtsZ ring formation and cell size in individual Escherichia coli bacteria. Upon inducing overexpression of minE, cell size increases gradually to a new steady-state value. Concurrently, the time required to initiate FtsZ ring formation grows as the size approaches the new steady-state, at which point the ring formation initiates as early as before induction. These results highlight the contribution of Min proteins to cell size control, which may be partially responsible for the size fluctuations observed in bacterial populations, and may clarify how the size difference acquired during asymmetric cell division is offset.

Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli.

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to determine the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells, which is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging.

Coordinated peptidoglycan synthases and hydrolases stabilize the bacterial cell wall

Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.

Control of the flagellation pattern in Helicobacter pylori by FlhF and FlhG.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and β-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Cell shape instability during cytokinesis in tetraploid HCT116 cells

Tetraploidy is a hallmark of broad cancer types, but it remains largely unknown which aspects of cellular processes are influenced by tetraploidization in human cells. Here, we found that tetraploid HCT116 cells manifested severe cell shape instability during cytokinesis, unlike their diploid counterparts. The cell shape instability accompanied the formation of protrusive deformation at the cell poles, indicating ectopic contractile activity of the cell cortex. While cytokinesis regulators such as RhoA and anillin correctly accumulated at the equatorial cortex, myosin II was over-accumulated at the cell poles, specifically in tetraploid cells. Suppression of myosin II activity by Y27632 treatment restored smooth cell shape in tetraploids during cytokinesis, indicating dysregulation of myosin II as a primary cause of the cell shape instability in the tetraploid state. Our results demonstrate a new aspect of the dynamic cellular process profoundly affected by tetraploidization in human cells, which provides a clue to molecular mechanisms of tetraploidy-driven pathogenic processes.

#3760 CYTOSKELETON-ORGANELLE INTERACTION IN LIVING CELL EXPRESSING INF2 VARIANTS CAUSING FSGS AND PERIPHERAL NEUROPATHY

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a common glomerular injury and represents a leading cause of ESRD. Studies with genetic FSGS show that a majority of genes are associated with regulation of podocyte cytoskeletons. INF2 is an actin assembly factor that mediates both polymerization and depolymerization and is the most frequent cause of dominant inherited FSGS. INF2 mutations located in the proximal half of DID (residues Leu57 to Glu184) typically cause CMT with early-onset FSGS, whereas that situating in the distal half (residues Glu184 to Leu245) cause FSGS alone. Previous studies disclosed that INF2 mutations primarily disorganize actin network and alter mitochondria shapes and vesicle motility. However, it remains elusive how the cytoskeletal network is concerted with the organelle morphology and functions in the disease cells. This study aims to characterize the interaction between cytoskeleton and organelle in cells expressing INF2 FSGS or CMT/FSGS variants. Method We characterized the cytoskeletal-organelle interaction in live HeLa or COS-7 cells expressing pathogenic INF2 variants in our cohort of FSGS. We performed a triple labeling by co-transfection of GFP-INF2, cytoskeletal markers (Lifeact-mCherry or EMTB-mScarletI), and by the uptake of organelle markers (Lysotracker or Mitotracker). To clarify the detailed pattern and dynamics of cytoskeletons and organelles, we employed the Spinning Disk Confocal Microscope DragonFly, which allows real-time super-resolution imaging with 70nm resolution. Results Wild-type-INF2 (WT-INF2) cells showed an ER pattern with perinuclear, Golgi accumulation. Mild INF2 variants causing FSGS alone (T161N, N202S) preserved the subcellular distribution essential similar to WT-INF2, while showing focal irregularity of ER networks. In contrast, severe INF2 variants leading to CMT/FSGS (G73D) exhibited a diffuse coarse ER pattern with few Golgi clusters and occasional peripheral accumulation in both cell poles. In live imaging of cells coexpressing GFP-INF2 and Lifeact-mCherry, actin stress fibers were pronouncedly reduced in CMT/FSGS variant (G73D) cells, but were mildly decreased FSGS variant (T161N and N202S) cells, compared with WT-INF2 cells. The filopodia in cells expressing T161N variant were less mobile (n = 10, P< 0.01) but were longer (n = 10, P< 0.0001) than those in cells expressing WT-INF2. T161N variant reduced expression of cortactin that stabilizes actin-branching and narrowed area of cortical actin meshwork compared to the WT-INF2 (n = 11, P< 0.01), suggesting that INF2 could serve the cortical actin formation. HeLa cells coexpressing T161N variant and EMTB showed a bipolar cell elongation along the longitudinal axis (n≥30, P< 0.0001) and disarranged microtubule bundles aligned in parallel, compared with those expressing WT-INF2. A time-lapse imaging of living COS-7 cells revealed that T161N variant increases mitochondrial fragmentation as well as mitochondria-ER interfaces more prominent than WT-INF2. Moreover, T161N variant induced the mitochondria misdistribution at the cell periphery (filopodia and cell edge) in both cell poles. Such aberrant patterns frequently coexisted with disarranged microtubules. Moreover, in lysotracker-labeled living COS-7, T161N variant reduced the motility of the lysosomal vesicles in cortical actin meshwork area, compared with WT-INF2 (n = 100, P< 0.0001). These observations suggest that INF2 variants perturbate the trafficking of organelles through disorganization of actin-microtubule network. Conclusion Our results indicate that INF2 CMT/FSGS variants cause more global and severe organelle dysfunction by disrupting the organelle-cytoskeletal interaction, than INF2 FSGS variants. The microtubule disarrangement might be due to an insufficient capping mechanism by which the microtubules are stably tethered to the cortical actin meshwork. Elucidation of cell-specific factors will help better understanding why podocyte and Schwann cell are susceptible to the cytoskeletal disarrangement.

The divisome but not the elongasome organizes capsule synthesis in Streptococcus pneumoniae

The bacterial cell envelope consists of multiple layers, including the peptidoglycan cell wall, one or two membranes, and often an external layer composed of capsular polysaccharides (CPS) or other components. How the synthesis of all these layers is precisely coordinated remains unclear. Here, we identify a mechanism that coordinates the synthesis of CPS and peptidoglycan in Streptococcus pneumoniae. We show that CPS synthesis initiates from the division septum and propagates along the long axis of the cell, organized by the tyrosine kinase system CpsCD. CpsC and the rest of the CPS synthesis complex are recruited to the septum by proteins associated with the divisome (a complex involved in septal peptidoglycan synthesis) but not the elongasome (involved in peripheral peptidoglycan synthesis). Assembly of the CPS complex starts with CpsCD, then CpsA and CpsH, the glycosyltransferases, and finally CpsJ. Remarkably, targeting CpsC to the cell pole is sufficient to reposition CPS synthesis, leading to diplococci that lack CPS at the septum. We propose that septal CPS synthesis is important for chain formation and complement evasion, thereby promoting bacterial survival inside the host.

Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Conjugation is a major form of horizontal gene transfer, contributing to bacterial evolution and the acquisition of new traits. During conjugation, a donor cell transfers DNA to a recipient through a specialized DNA translocation channel classified as a type IV secretion system (T4SS). Here, we focused on the T4SS of ICEBs1, an integrative and conjugative element in Bacillus subtilis. ConE, encoded by ICEBs1, is a member of the VirB4 family of ATPases, the most conserved component of T4SSs. ConE is required for conjugation and localizes to the cell membrane, predominantly at the cell poles. In addition to Walker A and B boxes, VirB4 homologs have conserved ATPase motifs C, D, and E. Here, we created alanine substitutions in five conserved residues within or near ATPase motifs in ConE. Mutations in all five residues drastically decreased conjugation frequency but did not affect ConE protein levels or localization, indicating that an intact ATPase domain is critical for DNA transfer. Purified ConE is largely monomeric with some oligomers and lacks enzymatic activity, suggesting that ATP hydrolysis may be regulated or require special solution conditions. Finally, we investigated which ICEBs1 T4SS components interact with ConE using a bacterial two-hybrid assay. ConE interacts with itself, ConB, and ConQ, but these interactions are not required to stabilize ConE protein levels and largely do not depend on conserved residues within the ATPase motifs of ConE. The structure-function characterization of ConE provides more insight into this conserved component shared by all T4SSs. IMPORTANCE Conjugation is a major form of horizontal gene transfer and involves the transfer of DNA from one bacterium to another through the conjugation machinery. Conjugation contributes to bacterial evolution by disseminating genes involved in antibiotic resistance, metabolism, and virulence. Here, we characterized ConE, a protein component of the conjugation machinery of the conjugative element ICEBs1 of the bacterium Bacillus subtilis. We found that mutations in the conserved ATPase motifs of ConE disrupt mating but do not alter ConE localization, self-interaction, or levels. We also explored which conjugation proteins ConE interacts with and whether these interactions contribute to stabilizing ConE. Our work contributes to the understanding of the conjugative machinery of Gram-positive bacteria.

In vivo imaging of MmpL transporters reveals distinct subcellular locations for export of mycolic acids and non-essential trehalose polyphleates in the mycobacterial outer membrane

The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled.

Interaction of FlhF, SRP-like GTPase with FliF, MS ring component assembling the initial structure of flagella in marine Vibrio.

Vibrio alginolyticus forms a single flagellum at its cell pole. FlhF and FlhG are known to be the main proteins responsible for the polar formation of single flagellum. MS-ring formation in the flagellar basal body appears to be an initiation step for flagellar assembly. The MS-ring is formed by a single protein, FliF, which has two transmembrane (TM) segments and a large periplasmic region. We had shown that FlhF was required for the polar localization of Vibrio FliF, and FlhF facilitated MS-ring formation when FliF was overexpressed in Escherichia coli cells. These results suggest that FlhF interacts with FliF to facilitate MS-ring formation. Here, we attempted to detect this interaction using Vibrio FliF fragments fused to a tag of Glutathione S-transferase in E. coli. We found that the N-terminal 108 residues of FliF, including the first TM segment and the periplasmic region, could pull FlhF down. In the first step, signal recognition particle (SRP) and its receptor are involved in the transport of membrane proteins to target them, which delivers them to the translocon. FlhF may have a similar or enhanced function as SRP, which binds to a region rich in hydrophobic residues.

Diving into the complexity of the spirochetal endoflagellum.

Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. Their cell morphology, subcellular organization, and metabolism reveal atypical features. Spirochetal motility is also singular, dependent on the presence of periplasmic flagella or endoflagella, inserted subterminally at cell poles and not penetrating the outer membrane and elongating outside the cell as in enterobacteria. In this review we present a comprehensive comparative genomics analysis of endoflagellar systems in spirochetes, highlighting recent findings on the flagellar basal body and filament. Continued progress in understanding the function and architecture of spirochetal flagella is uncovering paradigm-shifting mechanisms of bacterial motility.

DivIVA Phosphorylation Affects Its Dynamics and Cell Cycle in Radioresistant Deinococcus radiodurans.

DivIVA is a member of the Min family of proteins that spatially regulates septum formation at the midcell position and cell pole determination in Bacillus subtilis. Deinococcus radiodurans, a Gram-positive coccus-shaped bacterium, is characterized by its extreme resistance to DNA-damaging agents, including radiation. D. radiodurans cells exposed to gamma radiation undergo cell division arrest by as-yet-uncharacterized mechanisms. divIVA is shown to be an essential cell division gene in this bacterium, and DivIVA of D. radiodurans (drDivIVA) interacts with genome segregation proteins through its N-terminal region. Earlier, RqkA, a gamma radiation-responsive Ser/Thr quinoprotein kinase, was characterized for its role in radioresistance in D. radiodurans. Here, we showed that RqkA phosphorylates drDivIVA at the threonine 19 (T19) residue. The phospho-mimetic mutant with a mutation of T19 to E19 in DivIVA (DivIVAT19E) is found to be functionally different from the phospho-ablative mutant (DivIVAT19A) or the wild-type drDivIVA. A DivIVAT19E-red fluorescent protein (RFP) fusion expressed in the wild-type background showed the arrest in the typical dynamics of drDivIVA and the loss of its interaction with the genome segregation protein ParA2. The allelic replacement of divIVA with divIVAT19E-rfp was not tolerated unless drDivIVA was expressed episomally, while there was no phenotypic change when the wild-type allele was replaced with either divIVAT19A-rfp or divIVA-rfp. These results suggested that the phosphorylation of T19 in drDivIVA by RqkA affected its in vivo functions, which may contribute to the cell cycle arrest in this bacterium. IMPORTANCE Deinococcus radiodurans, a radioresistant bacterium, lacks LexA/RecA-mediated DNA damage response and cell cycle regulation as known in other bacteria. However, it adjusts its transcriptome and proteome upon DNA damage. In eukaryotes, the DNA damage response and cell cycle are regulated by Ser/Thr protein kinases. In D. radiodurans, we characterized a gamma radiation-responsive Ser/Thr quinoprotein kinase (RqkA) that phosphorylated DNA repair and cell division proteins in this bacterium. In previous work, the effect of S/T phosphorylation by RqkA on activity improvement of the DNA repair proteins has been demonstrated. This study reports that Ser phosphorylation by RqkA attenuates the function of a cell polarity and plane of cell division-determining protein, DivIVA, and its cellular dynamics in response to DNA damage, which might help to understand the mechanism of cell cycle regulation in this bacterium.

Polarized branched Actin modulates cortical mechanics to produce unequal-size daughters during asymmetric division

The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

Optical imaging of synaptic activity in auditory hair cells.

The auditory system plays a vital role in living organisms, allowing them to navigate in space, communicate and avoid danger. Hair cells of the auditory and vestibular systems are capable of detecting sounds that induce sub nanometer vibrations of a collection of stereocilia that protrude from the apical surface of the cell body. Upon deflection of the hair bundle, tension increases in the tip links that connect adjacent pairs of stereocilia, which triggers a cascade of cellular events. Mechano-sensitive transduction channels that are coupled to the tip links enable the influx of ionic currents, leading to depolarization of the hair cell. As a consequence, neurotransmitters are released at the afferent synapse, generating spike trains in the neurons that ultimately convey signals to the brain. We studied the dynamics of afferent neurons from the sacculus of the American bullfrog (Rana catesbeiana) by means of fluorescence microscopy. Introducing direct mechanical stimulation of the hair bundle, as well as chemical manipulation of the immersion medium of the cells, allowed us to observe resulting changes in the light intensity dynamics emitted by fluorescent Ca2+ indicators. Spatial and temporal observations of Ca2+ related fluorescence signals were performed surrounding the basal pole of the hair cells in order to elucidate possible neuronal activation elicited by hair bundle activity.

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis.

Mycobacteria expand their cell walls at the cell poles in a manner that is not well described at the molecular level. In this study, we identify a new polar factor, PlrA, that is involved in restricting peptidoglycan metabolism to the cell poles in Mycobacterium smegmatis. We establish that only the N-terminal membrane domain of PlrA is essential. We show that depletion of plrA pheno-copies depletion of polar growth factor Wag31, and that PlrA is involved in regulating the Wag31 polar foci.

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum.

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole or how it is organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 helps to control proper septation as well as new and old pole elongation. We have identified key amino acid residues involved in these essential functions. Enzyme assays revealed that Wag31 interacts with lipid metabolism by modulating acyl-CoA carboxylase (ACCase) activity. We show that Wag31 does not control polar growth by regulating the localization of cell wall precursor enzymes to the Intracellular Membrane Domain, and we also demonstrate that phosphorylation of Wag31 does not substantively regulate peptidoglycan metabolism. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the need for new molecular models of Wag31 function.

Polarity of c-di-GMP synthesis and degradation.

The bacterial cell pole has long been recognized as a defined compartment for enzymatic activities that are important or even vital for the cell. Polarity of diguanylate cyclases and phosphodiesterases, enzymes that synthesize and degrade the second messenger c-di-GMP, has now been demonstrated for several bacterial systems. Here we review these polar regulatory systems and show how the asymmetry of c-di-GMP production and turnover in concert with different modes of activation and deactivation creates heterogeneity in cellular c-di-GMP levels. We highlight how this heterogeneity generates a diverse set of phenotypic identities or states and how this may benefit the cell population, and we discuss reasons why the polarity of c-di-GMP signaling is probably widespread among bacteria.