Switching of the Bacterial Flagellar Motor Near Zero Load

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW → CCW and CCW → CW switching rates increased linearly with motor torque. Evidently, the switch senses stator-rotor interactions as well as the CheY-P concentration.     

Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane

The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a DeltafliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.     

Nucleation and growth of cadherin adhesions

Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.     

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.     

基于激光共聚焦同步双扫描技术的LC3脱乙酰化修饰调控自噬的机理研究

激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。     

Visualizing HIV-1 Assembly

The assembly of an HIV-1 particle is a complex, multistep process involving several viral and cellular proteins, RNAs and lipids. While many macroscopic and fixed-cell microscopic techniques have provided important insights into the structure of HIV-1 particles and the mechanisms by which they assemble, analysis of individual particles and their assembly in living cells offers the potential of surmounting many of the limitations inherent in other approaches. In this review, we discuss how the recent application of live-cell microscopic imaging techniques has increased our understanding of the process of HIV-1 particle assembly. In particular, we focus on recent studies that have employed total internal reflection fluorescence microscopy and other single-virion imaging techniques in live cells. These approaches have illuminated the dynamics of Gag protein assembly, viral RNA packaging and ESCRT (endosomal sorting complex required for transport) protein recruitment at the level of individual viral particles. Overall, the particular advantages of individual particle imaging in living cells have yielded findings that would have been difficult or impossible to obtain using macroscopic or fixed-cell microscopic techniques.     

Subunit Exchange in Protein Complexes

Over the past 50?years, protein complexes have been studied with techniques such as X-ray crystallography and electron microscopy, generating images which although detailed are static and homogeneous. More recently, limited application of in vivo fluorescence and other techniques has revealed that many complexes previously thought stable and compositionally uniform are dynamically variable, continually exchanging components with a freely circulating pool of “spares.” Here, we consider the purpose and prevalence of protein exchange, first reviewing the ongoing story of exchange in the bacterial flagella motor, before surveying reports of exchange in complexes across all domains of life, together highlighting great diversity in timescales and functions. Finally, we put this in the context of high-throughput proteomic studies which hint that exchange might be the norm, rather than an exception.     

Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell

The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.     

Bi-directional transport of GLUT4 vesicles near the plasma membrane of primary rat adipocytes

Insulin stimulates glucose uptake into adipocytes by mobilizing intracellular membrane vesicles containing GLUT4 proteins to the plasma membrane. Here we applied time-lapse total internal reflection fluorescence microscopy to study moving parameters and characters of exogenously expressed GLUT4 vesicles in basal, insulin and nocodazole treated primary rat adipocytes. Our results showed that microtubules were essential for long-range transport of GLUT4 vesicles but not obligatory for GLUT4 distribution in rat adipocytes. Insulin reduced the mobility of the vesicles, made them tethered/docked to the PM and finally had constitutive exocytosis. Moreover, long-range bi-directional movements of GLUT4 vesicles were visualized for the first time by TIRFM. It is likely that there are interactions between insulin signaling and microtubules, to regulating GLUT4 translocation in rat adipocytes.     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

Model of chromosome motility in Drosophila embryos: adaptation of a general mechanism for rapid mitosis

During mitosis, ensembles of dynamic MTs and motors exert forces that coordinate chromosome segregation. Typically, chromosomes align at the metaphase spindle equator where they oscillate along the pole-pole axis before disjoining and moving poleward during anaphase A, but spindles in different cell types display differences in MT dynamicity, in the amplitude of chromosome oscillations and in rates of chromatid-to-pole motion. Drosophila embryonic mitotic spindles, for example, display remarkably dynamic MTs, barely detectable metaphase chromosome oscillations, and a rapid rate of "flux-pacman-dependent" anaphase chromatid-to-pole motility. Here we develop a force-balance model that describes Drosophila embryo chromosome motility in terms of a balance of forces acting on kinetochores and kMTs that is generated by multiple polymer ratchets and mitotic motors coupled to tension-dependent kMT dynamics. The model shows that i), multiple MTs displaying high dynamic instability can drive steady and rapid chromosome motion; ii), chromosome motility during metaphase and anaphase A can be described by a single mechanism; iii), high kinetochore dynein activity is deployed to dampen metaphase oscillations, to augment the basic flux-pacman mechanism, and to drive rapid anaphase A; iv), modulation of the MT rescue frequency by the kinetochore-associated kinesin-13 depolymerase promotes metaphase chromosome oscillations; and v), this basic mechanism can be adapted to a broad range of spindles.     

The C-terminal domain of the Arabidopsis AtMBD7 protein confers strong chromatin binding activity

The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.     

Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope

Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and beta-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP+/+ and MVP-/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus.     

Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains

Supported cell-membrane sheets are promising in vitro systems to investigate the properties of membranes with native protein/lipid composition, in particular their sub-compartmentalization and the differential localization of proteins associated to them. While such studies are usually performed using static microscopy techniques, we demonstrate here the potential offered by dynamic diffusion measurements. Whereas the overall fluidity of the lipid bilayer was preserved, the preparation of the membrane sheets led to the selective immobilization of extracellular and transmembrane (TM) glycosylated proteins and the anchored proteins/lipids associated with them. Taking advantage of this, we investigated the association of the G protein Gq with TM proteins, in particular G-protein coupled receptors (GPCRs), by monitoring the changes in diffusion occurring after preparation of the supported membranes. Two fluorescently tagged Galphaq proteins were constructed, which remained either mostly monomeric in the plasma membrane or associated with Gbetagamma in heterotrimers. While both constructs diffused similarly in living cells, the preparation of the supported membranes led to the selective immobilization of the heterotrimers with minimal changes of the diffusion of the monomeric Galphaq. The diverse mobility of monomeric and heterotrimeric Galphaq was a result of their different lipid anchors as demonstrated by monitoring the diffusion of the corresponding anchors alone. We propose that the immobilization of the heterotrimer was caused by its partitioning inside membrane microdomains surrounding GPCRs.     

Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer

To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 Å. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.     

Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 Å, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 Å. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5′-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.     

Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.     

Regulation of focal adhesion formation and filopodia extension by the cellular prion protein (PrPC)

While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell-substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA-like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src.     

Mechanism of the Ca-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca²⁺ binds to the EF2 domain of S100A4 with micromolar affinity and that the K_d value for Ca²⁺ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K_d results from a reduced dissociation rate constant (from 16 s^− 1 to 0.3 s^− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca²⁺]. However, for Ca²⁺ transients to be effective in further promoting dissociation, the elevated Ca²⁺ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.     

Molecular mechanism of bundle formation by the bacterial actin ParM

The actin homolog ParM plays a microtubule-like role in segregating DNA prior to bacterial cell division. Fluorescence and cryo-electron microscopy have shown that ParM forms filament bundles between separating DNA plasmids in vivo. Given the lack of ParM bundling proteins it remains unknown how ParM bundles form at the molecular level. Here we show using time-lapse TIRF microscopy, under in vitro molecular crowding conditions, that ParM-bundle formation consists of two distinct phases. At the onset of polymerization bundle thickness and shape are determined in the form of nuclei of short helically disordered filaments arranged in a liquid-like lattice. These nuclei then undergo an elongation phase whereby they rapidly increase in length. At steady state, ParM bundles fuse into one single large aggregate. This behavior had been predicted by theory but has not been observed for any other cytomotive biopolymer, including F-actin. We employed electron micrographs of ParM rafts, which are 2-D analogs of 3-D bundles, to identify the main molecular interfilament contacts within these suprastructures. The interface between filaments is similar for both parallel and anti-parallel orientations and the distribution of filament polarity is random within a bundle. We suggest that the interfilament interactions are not due to the interactions of specific residues but rather to long-range, counter ion mediated, electrostatic attractive forces. A randomly oriented bundle ensures that the assembly is rigid and that DNA may be captured with equal efficiency at both ends of the bundle via the ParR binding protein.