Chunlin Lu

..I present arguments here that ParM may be an apolar filament, in which the two helical strands are antiparallel...

..A combination of hydrodynamics and electron microscopy is especially powerful...

..Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation...

..We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli...

..A model is proposed here for how membrane-bound ZipA can reach around the FtsZ protofilament to bind the carboxy-terminal peptide, which faces away from the membrane...

..Finally, I address recent models that try to explain how protofilaments 1-subunit-thick show a cooperative assembly...

..The highly conserved amino acids are not those forming the subunit core or protofilament interface, but those involved in binding and hydrolysis of GTP...

..The second proposes that molecules in fibrils are already extended, and stretching is produced by force-induced unfolding of FN type III domains. Experimental observations that may help distinguish these two possibilities are discussed...

..This article discusses these findings and the molecular mechanisms that can now be addressed with the atomic structures...

..Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro...

..It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolar K(D)...

..Our study resolves conflicting interpretations from previous electron microscopy studies of L1...

..The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins...

..One ficolin gene in all species is expressed in liver and is the primary source of plasma ficolin. Expression of this gene in other tissues, and expression of the second ficolin gene, appears to vary in different species...

..We also demonstrate that Mre11 binds as a dimer between the catalytic domains of Rad50, bringing the nuclease activities of Mre11 in close proximity to the ATPase and DNA binding activities of Rad50...

..Immunoprecipitation with a 160-kDa kinectin-specific antibody brought down the 120-kDa kinectin. Thus, we suggest that kinectin is an extended parallel coiled-coil dimer, often a heterodimer...

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..Together our results implicate human U251 MG glial laminin as a previously uncharacterized laminin isoform with strong adhesive activity for fibroblasts and glial cells...

..We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied...

..7 was 42 s for MtbFtsZ compared with 5.5 s for EcFtsZ. Photobleaching studies in vivo showed a range of turnover half-times with an average of 25 s for MtbFtsZ as compared with 9 s for EcFtsZ...

..The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling...

..Our results thus favor an alternative model, which invokes a conformational change from a compact to an extended conformation, as the basis for FN fibril elasticity...

..Directional assembly could occur by either a treadmilling or a dynamic instability mechanism...

..The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix...

..This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model...

..Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?..

..This finding implies that assembly dynamics are determined primarily by GTP hydrolysis. Despite the greatly reduced assembly dynamics, the ftsZ84 cells divide with a normal cell-cycle time...

..When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched...

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..We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides...

..These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites...

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..We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers...

..This suggests that the TubZ polymers have a capping mechanism that may be related to the GTP cap that produces dynamic instability of microtubules...

..In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division...

..Finally, analysis of the heptad repeat shows that the a and d positions are more constrained in spacer rods, and the bcefg positions more constrained in skeletal muscle myosin...

..The mechanism may be related to the dynamic instability of microtubules...

..37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4-1.0 microM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism...

..Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation...

..The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation...

..Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton...

..Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification...

..The ITC data are highly suggestive of a cooperative assembly, although this is difficult to reconcile with the 1-subunit-thick protofilaments observed by electron microscopy...

..Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions...

..After our initial applications and testing in FN, these sensors should be widely applicable to determine the tension in other ECM and cytoskeletal systems. ..

..We will use high concentrations of the independently folding N-and C-terminal domains, to determine how they poison pf assembly and dynamics. We will also attempt to re-create the dimer nucleus from these domains. ..

..After our initial applications and testing in FN, these sensors should be widely applicable to determine the tension in other ECM and cytoskeletal systems. ..

..It also has potential clinical relevance. FtsZ is highly conserved in bacteria, and is an attractive target for new antibiotics. Several lead compounds targeting FtsZ are already being studied and developed. ..

..We will use high concentrations of the independently folding N-and C-terminal domains, to determine how they poison pf assembly and dynamics. We will also attempt to re-create the dimer nucleus from these domains. ..

..Overall, we are aiming for a complete characterization of the biophysics of FtsZ protofilament assembly in vitro, and complementary analysis by FRAP of assembly dynamics and function in vivo. ..

..5 if the knockout is from the beginning). FN is expressed during the development of many organs after E10, so our timed knockout should demonstrate where and how FN is important in organ development. ..

..We will use a combination light scattering and electron microscopy to characterize small polymers of these two proteins, and especially to investigate the question of nucleation of polymerization...

..Projects for this module are proposed by two faculty from Cell Biology and two from Biochemistry. This will be the only FCS facility at Duke University, and we expect additional projects from across the campus. ..

..It also has potential clinical relevance. FtsZ is highly conserved in bacteria, and is an attractive target for new antibiotics. Several lead compounds targeting FtsZ are already being studied and developed. ..