Oligomerization and higher‐order assembly contribute to sub‐cellular localization of a bacterial scaffold

In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub‐cellular localization and recruitment activity. We identified a domain within the C‐terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N‐terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C‐terminal domain also blocked discrete steps in the assembly of higher‐order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self‐associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub‐cellular localization. Thus, PopZ undergoes multiple orders of self‐assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.     

A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole

Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.     

Spatiotemporal control of PopZ localization through cell cycle–coupled multimerization

Bacterial cell poles constitute defined subcellular domains where numerous proteins localize, often at specific times, to affect various physiological processes. How pole recognition occurs and what governs the timing of protein localization are often unknown. In this paper, we investigate the mechanisms governing the localization of PopZ, a chromosome-anchoring protein whose unipolar to bipolar localization pattern is critical for cell cycle progression in Caulobacter crescentus. We provide evidence that polar localization of PopZ relied on its self-assembly into a higher-order structure (matrix) and that the unipolar to bipolar transition was coupled to the asymmetric distribution of ParA during the translocation of the origin-proximal ParB–parS partition complex. Collectively, our data suggest a model in which a local increase of ParA concentration promotes the assembly of a PopZ matrix precisely when and where this matrix is needed. Such coupling of protein assembly with a cell cycle–associated molecular asymmetry may represent a principle of cellular organization for controlling protein localization in both time and space.     

SpbR overproduction reveals the importance of proteolytic degradation for cell pole development and chromosome segregation in Caulobacter crescentus

In most rod‐shaped bacteria, DNA replication is quickly followed by chromosome segregation, when one of the newly duplicated centromeres moves across the cell to the opposite (or ‘new’) pole. Two proteins in Caulobacter crescentus, PopZ and TipN, provide directional cues at the new pole that guide the translocating chromosome to its destination. We show that centromere translocation can be inhibited by an evolutionarily conserved pole‐localized protein that we have named SpbR. When overproduced, SpbR exhibits aberrant accumulation at the old pole, where it physically interacts with PopZ. This prevents the relocation of PopZ to the new pole, thereby eliminating a positional cue for centromere translocation. Consistent with this, the centromere translocation phenotype of SpbR overproducing cells is strongly enhanced in a ?tipN mutant background. We find that pole‐localized SpbR is normally cleared by ClpXP‐mediated proteolysis before the time of chromosome segregation, indicating that SpbR turnover is part of the cell cycle‐dependent program of polar development. This work demonstrates the importance of proteolysis as a housekeeping activity that removes outgoing factors from the developing cell pole, and provides an example of a substrate that can inhibit polar functions if it is insufficiently cleared.     

A self-associating protein critical for chromosome attachment, division, and polar organization in caulobacter

Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB*ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism.     

Functional Analysis of the Integrator Subunit 12 Identifies a Microdomain That Mediates Activation of the Drosophila Integrator Complex

The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3′ ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3′ end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3′ end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3′ end formation of Drosophila snRNA.     

Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy

Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength, etc., which influences the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date.Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77 K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.     

Functional anatomy of fusulinids (foraminifera): Significance of the polar torsion illustrated inTriticites andSchwagerina (Schwagerinidae)

The phenomenon of polar torsion is described in the fusulinid generaTriticites andSchwagerina. It is known in elongate extinct and extant alveolinids, but never has been mentioned in fusulinids. In the latter, polar torsion occurs in ellipsoidal to cylindrical forms with folded septa in the polar region as well as throughout. In analogy to in situ observations on extant alveolinids, the functional significance of the polar torsion is proposed to be related to the distribution of septal pores and the special arrangement of septal folds in the polar region.     

NMR solution structure of human cannabinoid receptor-1 helix 7/8 peptide: Candidate electrostatic interactions and microdomain formation

We report the NMR solution structure of a synthetic 40-mer (T³⁷⁷-E⁴¹⁶) that encompasses human cannabinoid receptor-1 (hCB1) transmembrane helix 7 (TMH7) and helix 8 (H8) [hCB1(TMH7/H8)] in 30% trifluoroethanol/H₂O. Structural features include, from the peptide’s amino terminus, a hydrophobic α-helix (TMH7); a loop-like, 11 residue segment featuring a pronounced Pro-kink within the conserved NPxxY motif; a short amphipathic α-helix (H8) orthogonal to TMH7 with cationic and hydrophobic amino-acid clusters; and an unstructured C-terminal end. The hCB1(TMH7/H8) NMR solution structure suggests multiple electrostatic amino-acid interactions, including an intrahelical H8 salt bridge and a hydrogen-bond network involving the peptide’s loop-like region. Potential cation-π and cation-phenolic OH interactions between Y³⁹⁷ in the TMH7 NPxxY motif and R⁴⁰⁵ in H8 are identified as candidate structural forces promoting interhelical microdomain formation. This microdomain may function as a flexible molecular hinge during ligand-induced hCB1 conformer transitions.     

Dynamics of the Bacterial Intermediate Filament Crescentin In Vitro and In Vivo

Background

Crescentin, the recently discovered bacterial intermediate filament protein, organizes into an extended filamentous structure that spans the length of the bacterium Caulobacter crescentus and plays a critical role in defining its curvature. The mechanism by which crescentin mediates cell curvature and whether crescentin filamentous structures are dynamic and/or polar are not fully understood.

Methodology/Principal Findings

Using light microscopy, electron microscopy and quantitative rheology, we investigated the mechanics and dynamics of crescentin structures. Live-cell microscopy reveals that crescentin forms structures in vivo that undergo slow remodeling. The exchange of subunits between these structures and a pool of unassembled subunits is slow during the life cycle of the cell however; in vitro assembly and gelation of C. crescentus crescentin structures are rapid. Moreover, crescentin forms filamentous structures that are elastic, solid-like, and, like other intermediate filaments, can recover a significant portion of their network elasticity after shear. The assembly efficiency of crescentin is largely unaffected by monovalent cations (K⁺, Na⁺), but is enhanced by divalent cations (Mg²⁺, Ca²⁺), suggesting that the assembly kinetics and micromechanics of crescentin depend on the valence of the ions present in solution.

Conclusions/Significance

These results indicate that crescentin forms filamentous structures that are elastic, labile, and stiff, and that their low dissociation rate from established structures controls the slow remodeling of crescentin in C. crescentus.     

Absence of metabolic cold adaptation and compensatory acclimation in the Antarctic fly, Belgica antarctica

The respiratory metabolism in larvae of the Antarctic fly, Belgica antarctica Jacobs (Diptera: Chironomidae) was investigated at Palmer Station, Anvers Island (64°46′S, 64°03′W). Oxygen consumption was linearly related to temperature from 0 to 20°C, respectively, 49 and 338 nl/mg live wt/hr. Maintenance at 0 and 10°C for 8 days had no differential effect on the metabolic rate, suggesting that larvae lack the ability for compensatory acclimation. A comparison of standard metabolism for polar and temperate chironomids revealed no elevation of metabolic rate in polar forms. However, polar species exhibited lower activation energies than temperate forms indicating that the respiratory metabolism of polar chironomids is relatively temperature independent.     

Changes in Cytokinins and Gibberellin-Like Substances in Pinus radiata Buds during Lateral Shoot Initiation and the Characterization of Ribosyl Zeatin and a Novel Ribosyl Zeatin Glycoside

Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of “short shoots” (needle fascicles) to “long shoots” (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.     

Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells

We investigated membrane targeting of the P2X₇ receptor (P2X₇R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein μ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X₇R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.

Structured summary

MINT-8055849:Beta-catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Osmotic stabilizer-coupled suppression of NDR defects is dependent on the calcium-calcineurin signaling cascade in Aspergillus nidulans

Establishment and maintenance of cell polarity are coordinated by signaling pathways such as NDR (nuclear Dbf2-related) protein-kinase signaling and calcium signaling pathway. The NDR family of kinase is structurally related to the human myotonic dystrophy kinase, which, when impaired, confers a disease that involves changes in cytoarchitecture and ion homeostasis. CotA kinase, a member of the NDR protein kinase family, forms a complex with MobB to regulate cell polarized growth in Aspergillus nidulans. Our previous study demonstrated that mobB/cotA defects could be suppressed by the osmotic stress in the presence of external calcium. In this study, via the genetic and molecular approach, we further demonstrated that Ca²⁺-permeable stretch-activated nonselective cation channel-MidA, calcium/calmodulin-dependent protein phosphatase catalatic subunit-CnaA and external calcium, but not voltage-gated calcium channel homolog-CchA, were required for the osmotic stabilizer-coupled suppression. The up-regulation of calcium/calcineurin signaling pathway induced by osmotic stress might be the reason for bypassing the requirements of NDR kinase complex, which is otherwise necessary for polar morphogenesis. Our results suggest that calcium-calcineurin signaling pathway coordinates with MobB/CotA kinase complex in regulating polarity growth via maintaining cellular calcium homeostasis. However, CchA may act differently as other components in calcium signaling pathway in Aspergillus nidulans. These findings provide an excellent opportunity to identify the potential pathway linking NDR protein-kinase network to calcium signaling pathway.     

Chromosome driven spatial patterning of proteins in bacteria

The spatial patterning of proteins in bacteria plays an important role in many processes, from cell division to chemotaxis. In the asymmetrically dividing bacteria Caulobacter crescentus, a scaffolding protein, PopZ, localizes to both poles and aids the differential patterning of proteins between mother and daughter cells during division. Polar patterning of misfolded proteins in Escherichia coli has also been shown, and likely plays an important role in cellular ageing. Recent experiments on both of the above systems suggest that the presence of chromosome free regions along with protein multimerization may be a mechanism for driving the polar localization of proteins. We have developed a simple physical model for protein localization using only these two driving mechanisms. Our model reproduces all the observed patterns of PopZ and misfolded protein localization--from diffuse, unipolar, and bipolar patterns and can also account for the observed patterns in a variety of mutants. The model also suggests new experiments to further test the role of the chromosome in driving protein patterning, and whether such a mechanism is responsible for helping to drive the differentiation of the cell poles.     

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK_a values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK_a values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK_a values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK_a was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N^ε2 state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK_a calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK_a values are governed by short-range polar and Coulomb interactions.     

The Legionella pneumophila GTPase Activating Protein LepB Accelerates Rab1 Deactivation by a Non-canonical Hydrolytic Mechanism

GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.     

Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins

What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back‐and‐forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole‐organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self‐organizing oscillator to create motion suitable for chromosome segregation.     

Roles for L o microdomains and ESCRT in ER stress-induced lipid droplet microautophagy in budding yeast

Microlipophagy (µLP), degradation of lipid droplets (LDs) by microautophagy, occurs by autophagosome-independent direct uptake of LDs at lysosomes/vacuoles in response to nutrient limitations and ER stressors in Saccharomyces cerevisiae. In nutrient-limited yeast, liquid-ordered (L_o) microdomains, sterol-rich raftlike regions in vacuolar membranes, are sites of membrane invagination during LD uptake. The endosome sorting complex required for transport (ESCRT) is required for sterol transport during L_o formation under these conditions. However, ESCRT has been implicated in mediating membrane invagination during µLP induced by ER stressors or the diauxic shift from glycolysis- to respiration-driven growth. Here we report that ER stress induced by lipid imbalance and other stressors induces L_o microdomain formation. This process is ESCRT independent and dependent on Niemann-Pick type C sterol transfer proteins. Inhibition of ESCRT or L_o microdomain formation partially inhibits lipid imbalance-induced µLP, while inhibition of both blocks this µLP. Finally, although the ER stressors dithiothreitol or tunicamycin induce L_o microdomains, µLP in response to these stressors is ESCRT dependent and L_o microdomain independent. Our findings reveal that L_o microdomain formation is a yeast stress response, and stress-induced L_o microdomain formation occurs by stressor-specific mechanisms. Moreover, ESCRT and L_o microdomains play functionally distinct roles in LD uptake during stress-induced µLP.