Unique Biochemical Features of the Cytokinetic Protein FtsZ of Mycobacteria

FtsZ, the bacterial cytokinetic protein, a structural homologue of mammalian β-tubulin, is present in bacteria of diverse genera, including mycobacteria. The FtsZ protein of Mycobacterium tuberculosis (M. tuberculosis FtsZ), the causative agent of tuberculosis, is the most studied among the mycobacterial FtsZ proteins as it is a potential anti-tuberculosis drug target. M. tuberculosis FtsZ possesses many unique biochemical features, which include slow polymerisation kinetics, presence of charged amino acids in the C-terminal domain that interacts with a variety of other cell division proteins, and the presence of specific amino acids at unique locations that makes it distinct from the FtsZ of other mycobacterial species and of other bacterial genera. On the other hand, although the FtsZ of Mycobacterium leprae (M. leprae FtsZ), the causative agent of leprosy, shows high level of conservation with M. tuberculosis FtsZ, it has biochemical properties that are very different from those of M. tuberculosis FtsZ due to the difference in specific amino acid residues at critical locations on the protein. The present review focuses on these structural features of M. tuberculosis FtsZ and M. leprae FtsZ, as studied by others and by us, in comparison to those of the FtsZ of other mycobacterial species and of other bacterial genera

In vitro polymerization of Mycobacterium leprae FtsZ OR Mycobacterium tuberculosis FtsZ is revived or abolished, respectively, by reciprocal mutation of a single residue

A single residue that dramatically influences polymerization of principal cell division protein FtsZ of Mycobacterium leprae (MlFtsZ) and Mycobacterium tuberculosis (MtFtsZ) has been identified. Soluble, recombinant MlFtsZ did not show polymerization in vitro, in contrast to MtFtsZ, which polymerised. Mutation of the lone non-conserved residue T172 in the N-terminal domain of MlFtsZ to A172, as it exists in MtFtsZ, showed dramatic polymerization of MlFtsZ–T172A in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in MlFtsZ, abolished polymerization of MtFtsZ–A172T in vitro. While T172A mutation enhanced weak GTPase activity of MlFtsZ, reciprocal A172T mutation marginally reduced GTPase activity of MtFtsZ in vitro. These observations demonstrate that the residue at position 172 plays critical role in the polymerization of MlFtsZ and MtFtsZ. A possible evolutionary correlation between the presence of polymerization-adversive or polymerization-favouring residue at position 172 in FtsZ and generation time of the respective bacterium are discussed

Theory of the unusual doping and temperature dependence of photoemission spectra in manganites

A recent, major, puzzle in the core-level photoemission spectra of doped manganites is the observation of a 1–2 eV wide shoulder with intensity varying with temperature T as the square of the magnetization over a T scale of order 200 K, an order of magnitude less than electronic energies. This is addressed and resolved here, by extending a recently proposed two-fluid polaron–mobile electron model for these systems to include core-hole effects. The position of the shoulder is found to be determined by Coulomb and Jahn-Teller energies, while its spectral weight is determined by the mobile electron energetics which is strongly T and doping dependent, due to annealed disorder scattering from the polarons and the t2g core spins. Our theory accounts quantitatively for the observed T dependence of the difference spectra, and furthermore, explains the observed correspondence between spectral changes due to increasing doping and decreasing T

The C-Terminally Truncated Mtftsz-DC169 Mutant of Mycobacterium Tuberculosis Ftsz Shows Gtpase and GTP-Induced, GTP-Specific Polymerization Activities in Vitro

We recently reported that the deletion mutant MtFtsZ-DC169 of Mycobacterium tuberculosis FtsZ (MtFtsZ), which lacked 169 C-terminal residues and retained only the first 210 residues, formed long polymers in about 30 s after the addition of GTP, like the FtsZ of Escherichia coli (EcFtsZ) (Anand et al., 2004). Both of these FtsZs were purified under denaturing conditions and refolded. By contrast, the full-length MtFtsZ, prepared under identical conditions of denaturation and refolding, formed equivalent-type long polymers in 10 min after the addition of GTP. These observations prompted us to suggest that the residues in the C-terminal region of the MtFtsZ protein might be responsible for imposing slow polymerization on MtFtsZ

A C-terminal deletion mutant of Mycobacterium tuberculosis FtsZ shows fast polymerization in vitro

The FtsZ protein forms a dynamic polymeric ring structure, which functions as the guiding scaffold for septal invagination at the mid-cell site during cytokinesis in bacterial cells (Bi & Lutkenhaus, 1991). FtsZ forms polymers in a GTP-dependent manner in vivo and under different cationic conditions in vitro (Andreu et al., 2002; Beuria et al., 2003;Bi & Lutkenhaus, 1991; Bramhill & Thompson, 1994; Caplan & Erickson, 2003; Diaz et al., 2001; Erickson et al., 1996; Lu et al., 1998; Mingorance et al., 2001; Mukherjee & Lutkenhaus, 1994, 1999; Rivas et al., 2000; Romberg et al., 2001; Scheffers et al., 2001; Wang et al., 1997; White et al., 2000; Yu & Margolin, 1997). The rate of polymerization of the FtsZ protein of Mycobacterium tuberculosis H37Rv (MtFtsZ) is remarkably slow when compared to that of the Escherichia coli FtsZ protein (EcFtsZ) (Bramhill & Thompson, 1994; Mukherjee & Lutkenhaus, 1999; White et al., 2000). While polymerization of EcFtsZ reaches steady state in 30 s after the addition of GTP in vitro (Bramhill & Thompson, 1994; Mukherjee & Lutkenhaus, 1999), MtFtsZ takes nearly 10 min to reach the same state (White et al., 2000). The rate of depolymerization of MtFtsZ was also found to be slow, in conformity with its slow GTPase activity (White et al., 2000)

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation--A Novel Role for NDK.

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented

Theory of the unusual doping and temperature dependence of photoemission spectra in manganites

A recent, major, puzzle in the core-level photoemission spectra of doped manganites is the observation of a 1-2 eV wide shoulder with intensity varying with temperature T as the square of the magnetization over a T scale of order 200 K, an order of magnitude less than electronic energies. This is addressed and resolved here, by extending a recently proposed two-fluid polaron-mobile electron model for these systems to include core-hole effects. The position of the shoulder is found to be determined by Coulomb and Jahn-Teller energies, while its spectral weight is determined by the mobile electron energetics which is strongly T and doping dependent, due to annealed disorder scattering from the polarons and the t<SUB>2g</SUB> core spins. Our theory accounts quantitatively for the observed T dependence of the difference spectra, and furthermore, explains the observed correspondence between spectral changes due to increasing doping and decreasing T

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

<div><p>Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.</p></div

Inhibition of Myo1 arrests cell division and affected blastomere shapeof Zebrafish embryos.

<p>Schematic representation of Myo1 domain structures. (A) Semiquantitative RT-PCR profiles from cDNA and RNA templates. For Myo1Ea&b, control (top panel) and with PClP (bottom panel), were compared at 1–4 and 64 cells stages for cDNA and RNA templates, (B) Semiquantitative RT-PCR profiles from cDNA and RNA templates. For Myo1Cb, control (top panel) and with PClP (bottom panel), were compared at 1–4 and 64 cells for cDNA and RNA templates. (C) (i) Western blot for Myo1C relative-levels at 1–4 and 64 cell stages. Control (top panel) and with PClP (bottom panel), GAPDH used as loading control. (ii) Relative change in levels of Myo1C ±PClP, 1–4 and 64 cell stages, control grey, PClP black. Error bars indicate SD, n = 3. (D) (i) Top panel, control embryos at different developmental stages. Bottom panel, PClP treated embryos taken in identical time as in control, dotted line shows boundary between yolk and blastodisc in both panels, (ii) measurement of changes in blastodisk thickness, as indicated by vertical both sided arrows in(Ei) with time, approximately along first cleavage furrow in both, the control (dotted line) and PClP treated embryos (solid line), n = 8, error indicate SD. Black arrow-head in time axis indicates PClP addition. Embryos were observed in lateral or side view position. (E) DAPI stained nucleus profile of 64-cell control (2 hpf) (top panel) and equivalent 2 hpf PClP inhibited 8- cell embryo (middle panel). 1 hpf control 8- cell embryo, showing divided nucleus is also shown (lower panel). bar 120μm in all places.</p