In this work, we identified and characterized developmentally regulated aspects to cell division in the Xenopus laevis. We found that cells in the early neural plate divide in an oriented manner. This orientation is established by Cdc42 controlled maintenance of stable interactions between the spindle and the cell cortex. This role of Cdc42 is developmentally regulated and cells dividing later in a related tissue, the tail epidermis, are not under this control. Moreover, we find that the cell divisions in the early neural plate are further specialized in their mechanisms of cell division. Cells in the early neural plate exhibit exaggerated anaphase-B movements, a delayed onset of cytokinesis, low density of midzone microtubules and a rapid cytokinetic furrow ingression as compared to the late tail epidermis, another ectodermally derived tissue. These modifications to the mechanism of cell division appear to be because of a reduced level of PRC1, a microtubule bundling protein, and thus modifications to the central spindle structure. Finally, we find that cytokinetic mechanisms may be functionally related to the process of ciliogenesis. We find proteins known to localize to the central spindle localized to the rootlet of the basal body of cilia in multiciliated cells of the mucociliary epidermis. This localization may be related to vesicle transport during both these processes. This work reveals unexpected plasticity to fundamental mechanisms of cell division. / text

FtsZ, the principal cell-division protein, polymerizes in GTP-dependent manner in vitro (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994; Rivas et al., 2000). FtsZ polymerization at the mid-cell site of bacterium leads to formation of a guiding scaffold, the Z-ring, for bacterial cytokinesis (Bi and Lutkenhaus, 1991; Sun and Margolin, 1998). GTP-induced polymerization process of FtsZ can be monitored in vitro Using 90º light scattering (Mukherjee and Lutkenhaus, 1999) and polymers formed can be visualized using transmission electron microscopy (Lu and Erickson, 1998) or quntitated in terms of the amount of FtsZ polymer pelleted during ultracentrifugation (Mukherjee and Lutkenhaus, 1998). The research work presented in this thesis focused on structure-function correlative analysis of Mycobacterium tuberculosis FtsZ(MtFtsZ0 and FtsZ proteins of Mycobacterium leprae (M1FtsZ), Mycobacterium smegmatis(MsFtsZ), and Streptomyces coelicolor (ScFtsZ) (as it is from Actinomycetes family to which mycobacteria belong) in vitro. It was initiated with investigation on the biochemical properties of Mycobacterium leprae FtsZ (M1FtsZ) in vitro. In comparison with those of MtFtsZ. Subsequently, the role of C-terminal stretch of amino acid residues of MtFtsZ in polymerization was investigated. Finally, a comparative analysis of the biochemical properties of MtFtsZ, MsFtsZ, and ScFtsZ was carried out in order to find out whether a correlation exists between the time taken by the FtsZ of a bacterium to polymerise and the generation time of the organism. The thesis is presented in five chapters. First Chapter gives an exhaustive introduction on the structure-function aspects of FtsZ. Second Chapter deals with materials used in this research work and details of various experimental methods [cloning and expression of FtsZ (White et. Al., 2000), decision and point mutagenesis, preparation of His-tag free MtFtsZ and M1FtsZ by thrombin cleavage method, 90º light scattering (Mukherjee and Lutkenhaus, 1999), White, et al., 2000), transmission electron microscopy (Lu and Erickson, 1998), pelleting assay for polymeric FtsZ (Mukherjee and Lutkenhaus, 1998), GTP-binding by UV-crosslinking (RayChaudhuri and Park, 1992; de Boer et al.,) GTPase assay(RayChaudhuri and Park, 1992); de Boer et al., 1992), Circular Dichroism (Saxena and Wetlaufer, 1971) and ANS fluorescence emission spectroscopy (Semisotnov, et al., 1991)]. The Chapters three to five contain all the data related to the research work, the outlines of which are given below. Chapter 3. Biochemical Characterisation of FtsZ Protein of Mycobacterium leprae In Comparison with the Biochemical Properties of FtsZ Protein of Mycobacteriulm tulberculosis In Vitro The major finding in this part of thesis work is on the demonstration that single reciprocal point mutation partially revives polymerization-inactive M1FtsZ and Inactivates polymerization-active MtFtsZ in vitro. In brief, soluble, recombinant M1FtsZ did not show detectable polymerization in vitro, in contrast to MtFtsZ, which showed appreciable polymerization, under standard conditions, when monitored using 90º light scattering assay and transmission electron microscopy. This was a surprising result, as M1FtsZ and MtFtsZ has 96% protein sequence identity. Mutation f T172 in the N-terminal domain of M1FtsZ to A172, as it exists in MtFtsZ, showed dramatic levels of polymerization in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in M1FtsZ, abolished polymerization in vitro. Further, M1FtsZ showed weak GTPase activity, in contrast to MtFtsZ, which showed appreciable GTPase activity. While T172A mutation enhanced GTPase activity of MtFtsZ in vitro. Circular dichroism spectroscopy and ANS fluorescence emission spectroscopy showed that there were no major secondary or tertiary structural changes in these point mutants. These observations demonstrate that the residue at position 172 plays a critical role in the polymerization of M1FtsZ and MtFtsZ, without appreciably affecting their respective GTpPase activity. Further, this result might have implications on evolution of a slow polymerizing FtsZ in slow growing bacteria. Further details of evolution related questions are addressed in Chapter 5. Chapter 4. Role of Carboxy Terminal Residues in the Biochemical Properties of FtsZ Protein of Mycobacterium tuberculosis In Vitro The major finding in this part of thesis work is the demonstration that the C-terminal end residues are critically required for polymerization of MtFtsZ in vitro, which is in direct contrast to the dispensability of C-terminal residues of Escherichia coli FtsZ(EcFtsZ), Bacillus subtilis FtsZ (BsFtsZ), and Pseudomonas aeruginosa (PaFtsZ) for polymerization. FtsZ protein from several bacterial species namely, Methanococcus jannaschii (MjFtsZ), Bacillus subtillis(BsFtsZ), Pseudomonas aeruginosa (PaFtsZ), and Aquifex aeolicus (AaFtsZ) (Lowe and Amos, 1998; Oliva et al., 2007), and Mycobacterium tuberculosis H37Rv (mtFtsZl Leung et al., 2004), whose crystal structures have been solved so far, were found to possess an N-terminal domain and a C-terminal domain that were connected to each other through a helix. The extreme C-terminal portion of all these FtsZ proteins is constituted by an unstructured tail (Lowe and Amos, 1998; Oliva et al., 2007l Leung et al., 2004), which is not found in the respective crystal structure of the protein. We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (mtFtsZ) have any role in MtFtsZ polymerization in vitro. Deletion of C-terminal 66 residues (313-379) was found to abolish polymerization. Replacement of the C-terminal 66 residues with the extreme C-terminal 13-residue stretch (DDDDVDVPPFMRR) did not restore polymerization. Although the terminal R in DDDDVDVPPFMRR is dispensable for full-length MtFtsZ polymerization, the terminal R in DDDDVDVPPFMR is indispensable for polymerization. Neither replacement of this R, in the terminal R deletion mutant DDDDVDVPPFMR, with K/H/D/A residues enabled polymerization. GTP binding and GTPase activities of the mutants were partially affected. The indispensable nature of C-terminal residues for MtFtsZ polymerization in vitro is contrary to the dispensability of the equivalent extreme C-terminal residues of Escherichi coli, Pseudomonas aeruginosa, and Bacillus subtilis FtsZ (Wang et. Al., 1997; Cordell et al., 2003; Singh et al., 2007) for in vitro polymerization. The essentiality of C-terminal extreme residues of BtFtsZ for polymerization offers direction to design anti MtFtsZ polymerization agents. Chapter 5. An attempt to find correlation between Biochemical properties of FtsZ and Generation Time of the Bacterium The clue that there might be a correlation between FtsZ polymeristion and generation time of the bacterium came from the observation mentioned in chapter 3. The presence of polymerization-aversive T172 in the FtsZ of extremely slow-growing M. leprae 913.5 days generation time, Levy, 1970) and polymerization-favouring A172 in the FtsZ of M. tuberculosis(18hrs generation time, Patterson and Youmans, 1970). For a bacterium, which has short generation time, it might be conducive to have an FtsZ that will also polymerise fast. Conversely, for a bacterium, which has long generation, it might be conducive to have an FtsZ molecule that will polymerise slow. In this respect, a preliminary comparative study was carried out between the generation time of bacterial species, E. coli, Mycobacterium smegmatis, Streptomyces coelicolor, M leprae, and M. tuhberculosis and their respective FtsZ (EcFtsZ, MsFtsZ, M1FtsZ and MtFtsZ). Detailed biochemical characterization of EcFtsZ and MtFtsZ has already been reported in the literature. In this thesis work, biochemical characterisation of M1FtsZ(Chapter 3), ScFtsZ and MsFtsZ (in this Chapter) were carried out. E. coli, which has a generation time of 18-55 min(labrum, 1953), possesses FtsZ (EcFtsZ) that reaches steady state of polymerization in about 10 sec under standard conditions in vitro (Beamhill and Thompson, 1994), using 90º light scattering assay (Mukherjee and Lukenhaus, 1999). On the other hand, M. tuberculosis, which has a generation time of 18hrs in vivo (Patterson and Youmans, 1970) and 24 hrs in vitro (Hiriyanna and Ramakrishnan, 1986) possesses FtsZ (MtFtsZ) that reaches steady state of polymerization in about 6 min post-addiction of GTP in vitro (White et al., 2000). Further, M. leprae, which takes 13.5 days tp divide once in vivo (levy, 1970), possesses an FtsZ (M1FtsZ) that does not even show polymerization under standard conditions in vitro (Chapter 3 of this thesis). The organisms Mycobacterium smegmatis and Streptomyces coelicolor have generation times that fall in between those of the other three organisms mentioned above. While M. smegmatis divides once in 2-3 hrs (Husson, 1998), S. coelicolor has a variable generation time depending on growth condition, which can be as fast as once in 2.31 hours, depending upon growth conditions (Cox, 2004). We found ScFtsZ and MsFtsZ takes around 4 min to reach polymerization saturation after addition of GTP, EcFtsZ( 10 sec), MtFtsZ (10 min) and M1FtsZ (dose not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation after addition of GTP, EcFtsZ (10sec), MtFtsZ (10 min) and M1FtsZ (does not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation time and the generation time of the respective bacterium. But when we compared polymerization time of ScFtsZ and MsFtsZ (4 min both case) with MtFtsZ ( 6 min), we found that there is no linear correlation with generation time of these bacteria and the time taken by their FtsZ to reach steady state of polymerization. Many more bacterial FtsZ proteins need to be characterized to conclusively state wthether there exist a correlation between generation time of bacteria and the time taken for their FtsZ to reach steady state of polymeristion. Such correlation would simply reveal the fact that the primary structure of an FtsZ protein might have evolved to suit the generation time of the bacterium.

En fin de cytocinèse, le fin pont cytoplasmique qui relie les deux cellules filles est clivé au niveau d’une structure dense en microtubules, le midbody, et permet ainsi la séparation physique de leurs deux cytoplasmes. Les mécanismes cellulaires et moléculaires de ce processus, appelé abscission, sont très étudiés dans des modèles de cellules en culture. Cependant, ils restent encore mal connus dans le contexte d’un organisme en développement. L’ovogenèse de drosophile est un modèle de choix pour l’étude de la régulation développementale de l’abscission. En effet, des cellules à abscission complète (cellules souches germinales) et incomplète (cystes germinaux) sont situées côte à côte au sein de la même unité développementale, le germarium. Les cellules souches se divisent asymétriquement, pour donner une autre cellule souche et un cystoblaste individualisé. Celui-ci entre en alors en différenciation, un programme au cours duquel il réalise quatre cycles cellulaires synchrones au cours desquels la cytocinèse est incomplète. Un cyste germinal de seize cellules interconnectées est ainsi formé. La durée de l’abscission est régulée précisément et dépend du contexte développemental. Notre laboratoire a récemment montré que les kinases Aurora B et Cdk1/ Cyclin B sont des régulateurs de la durée d’abscission dans les cellules germinales de drosophile et en cellules en culture de vertébrés. Mon travail a consisté à explorer la fonction de la protéine Shrub, un membre du complexe ESCRT-III, au cours de l’abscission dans la lignée germinale femelle de drosophile. Nous avons montré que Shrb est localisé au midbody des cellules souches en fin de cytocinèse, et promeut l’abscission. En effet, nous avons montré qu’une réduction du niveau de Shrub dans la lignée germinale provoque un fort délai de l’abscission des cellules souches, supérieur à la durée de leur cycle cellulaire. La cellule souche et son cystoblaste restent donc connecté jusqu’à la mitose suivante, formant ainsi des structures de plusieurs cellules connectées, appelées stem-cyst . L’abscission tardive au sein du stem cyst libère un progéniteur binucléé qui entre en différenciation. En conséquence, des chambres ovariennes à 32 cellules, au lieu de 16, sont formées. De plus, la fonction de Shrub dans l’abscission semble être contrecarrée par Aurora B, puisqu’une réduction des niveaux d’Aurora B dans des hétérozygotes Shrub réduit le nombre de stem-cysts et de chambres à 32 cellules observés. Enfin, nous avons identifié un nouveau facteur impliqué lors de l’abscission, la protéine Lethal giant discs (lgd), dont la perte de fonction induit, comme celle de Shrub, la formation de stem-cysts. En accord avec son rôle dans l’abscission, nous avons montré que Lgd est localisé au midbody. Lgd est requis pour la fonction de Shrub dans la voie endosomale, mais son implication lors de la cytocinèse était inconnue. Nous avons montré qu’un niveau réduit de Lgd augmente le nombre de stem-cysts des hétérozygotes Shrub, indiquant que Lgd et Shrub fonctionnent ensemble pour l’abscission des cellules souches. De façon surprenante, un nombre réduit de chambres à 32 cellules est observé dans ces ovaires, suggérant une fonction antagoniste de Lgd sur Shrub dans les cystes germinaux. Dans ces cystes, une abscission tardive se produit, qui divise en deux cystes de 16 cellules les cystes de 32 cellules, et expliquant ainsi le paradoxe observé (plus de stem-cysts, mais moins de chambres à 32 cellules). / At the end of cytokinesis, a thin cytoplasmic intercellular bridge is cleaved to allow physical separation of the two daughter cells. This process is called abscission, and its cellular and molecular events have been extensively explored in yeast and isolated mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood.Drosophila oogenesis is a great model to study how abscission is regulated developmentally, as within the same developmental unit, the germarium, we find cells undergoing abscission next to others where this process is blocked. Indeed, the germline stem cell (GSC) divides asymmetrically to give rise to another GSC and to an individualized cystoblast. This cell then enters a well-studied process of differentiation, where through four rounds of mitosis with incomplete cytokinesis, gives rives to a cyst of 16 interconnected cells. The duration of abscission, seems to be tightly regulated and dependent on the developmental context. Our lab has recently discovered that AurB and CycB/Cdk1 function as abscission timers in Drosophila GSC and isolated mammalian cells. Thus, my work consisted in exploring how this process is regulated in the Drosophila female germline.We showed that the ESCRT-III protein Shrb localizes to the midbody of the dividing GSC, functioning to promote abscission. Indeed, we found that reduced levels of Shrb resulted in the blockage, or strong delay, of abscission in the GSC and formation of a structure similar to a cyst. In these so called stem-cysts, the GSC keeps dividing while interconnected to its daughter cells. As a consequence, we saw the appearance of egg chambers formed of 32 cells, instead of 16. Furthermore, Shrb function in abscission seems to be counteracted by AurB, as reducing AurB levels in Shrb heterozygous resulted in decreased stem-cysts and 32-cell cysts. Finally, Lethal giant discs (lgd), required for Shrb function in the endosomal pathway, was also seen localizing at the midbody and regulating abscission in GSCs. Removing one copy of Lgd from Shrb heterozygous increased the number of stem-cysts, but surprisingly the number of 32-cell cysts was reduced. This paradoxical result was explained with the observation of late abscission events in mitotic cysts, which divided the 32-cell cysts in the middle, leading to the formation of two cysts of 16 cells.

L’émergence de comportements collectifs cellulaires n’est pas bien comprise. Nous l’abordons dans deux systèmes biologiques. A l'échelle du micromètre lors de la constriction de l’anneau cytokinétique, nous montrons que des complexes d’acto-myosine s’auto-organisent sous forme d’agrégats dans la levure à fission et dans la cellule de mammifères. Ces auto-organisations découlent de règles d'interactions communes mais pour des fonctions distinctes, le transport et la génération de stress respectivement. A l'échelle de 100 micromètres, nous observons des pulsations corrélées de cellules épithéliales. Nous montrons les rôles du frottement avec la surface, et le couplage entre l’aire cellulaire, sa hauteur et sa contractilité. Nous présentons aussi deux études, des polyamines synthétiques pour étudier la polymérisation d'actine in vivo, puis l’inversion de sens dans la migration - la ratchetaxie. Cette thèse illustre l'importance des phénomènes physiques dans la dynamique cellulaire. / The emergence of collective behavior from the interaction of individual units is not clear. In this thesis, we address this question in two different systems at different scales. At the micrometer scale during cytokinetic ring constriction, we show that acto-myosin self-organizes into rotating and static clusters in fission yeast and mammalian cells. These self-organizations arise from common interaction rules, but to serve distinct functions, transport and stress generation respectively. At 100 micrometers scale, we report correlated pulsations of cells in an epithelial monolayer. We show the key roles of substrate friction, and the tight coupling between cell area, cell height and contractility. We also present two other studies: synthetic polyamines for studying actin polymerization in vivo, and direction reversal in single cell migration during ratchetaxis. Altogether, this PhD illustrates the importance of physical phenomena in cellular dynamics.

Les structures d'acto-myosine sont impliquées dans de nombreuses fonctions cellulaires. Comprendre leur organisation et leur comportement collectif est toujours difficile. Nous avons étudié l'anneau cytokinétique dans les cellules de mammifères et dans les levures de fission, en orientant les cellules dans les microcavités, ce qui permet de voir l'anneau dans un seul plan focal. Avec cette configuration, nous révélons de nouvelles structures et des dynamiques distinctes pour les deux systèmes cellulaires. Dans les cellules de mammifères, nous trouvons des motifs réguliers de la myosine et la formine. Les caractéristiques de ces motifs sont stables tout au long de sa fermeture et leur apparition coïncide avec la constriction. Nous proposons que ce phénomène est une propriété inhérente du réseau d'acto-myosine et que la formation de ces motifs entraîne une augmentation du stress. Ces hypothèses sont confirmées par notre modèle en champ moyen. Par contraste, l'anneau de levure de fission montre des inhomogénéités tournantes de l'actine, de la myosine, des protéines de la construction de la paroi (Bgs) et d'autres protéines. La dynamique des inhomogénéités de myosine est inchangée, si la croissance de la paroi est inhibée. Cependant, l'inhibition du mouvement des inhomogénéités conduit à l'arrêt de la fermeture. Nous proposons que la fermeture de l'anneau est entraînée par la rotation de l'actine et de la myosine qui tirent des protéines Bgs, lesquelles construisent ainsi le septum. Cette hypothèse est confirmée par nos calculs et par des simulations numériques. Nous suggérons que la transition entre les états de différents ordres et dynamiques pourrait être une façon de réguler in vivo les systèmes d'acto-myosine. / Actomyosin structures are involved in many cell functions. Understanding their organization and collective behavior is still challenging. We study the cytokinetic ring in mammalian cells and in fission yeasts, by orienting cells in microcavities. This allows seeing the ring in a single plane of focus. With this setup, we reveal new structures and distinct dynamics for both cellular systems. In mammalian cells we find a pattern of regular clusters of myosin and formin. The characteristics of this pattern are stable throughout closure and its formation coincides with the onset of constriction. We propose that its characteristic is an inherent property of the actomyosin network and that its formation leads to an increase in stress generation. These hypotheses are supported by our theoretical mean field model. In contrast, fission yeast rings show rotating inhomogeneities (speckles), i.e. rotations of actin, myosin, cell wall building proteins (Bgs) and other proteins. Myosin speckles dynamic is unchanged, if wall growth is inhibited. However, the inhibition of speckle motion leads to stalled closure. We propose that the ring closure is driven by the rotation of actin and myosin, which pull Bgs thereby building the septum. This model is supported by our calculations and by numerical simulations. We suggest that the transition between states of different orders and dynamics might be a way to regulate actomyosin systems in vivo.

The PhD thesis is on the studsy of the influence of the ftsZ antisense RNA and FtsH protease on the synthesis and function of the Escherichia coli cytokinetic protein, FtsZ, which mediates septation during cell division. Thus, it involves three molecules, FtsZ, ftsZ antisense RNA, and FtsH protease. While the E. coli ftsZ antisense RNA is being identified and structurally and functionally characterised for the first time, there has been some earlier studies in the laboratory in which the FtsH protease was found to have influence on the presence of the FtsZ rings at the mid-cell site. The Chapter 1 is the Introduction to the thesis presented in 3 parts –Part 1A, 1B, and 1C, introducing FtsZ and bacterial cell division, bacterial antisense RNAs, and FtsH protease, respectively. The Chapter 2 gives the description of the Materials and Methods used in the study. The Chapter 3 presents the identification, structural and functional characterisation of the ftsZ cis-antisense RNA, and its role in the regulation of FtsZ protein levels. Initially, the expression of cis-encoded antisense RNA from E. coli ftsZ loci was demonstrated during the different growth phases of the bacterium (RT-PCR/qPCR data). Antisense RNA is expressed from three promoters (primer extension and promoter probe data) on the complementary strand of the ftsZ coding region and terminates at the singletrand te complementary toftsAthegenethat 3’islocatedregionupstreamof theofftsZ the gene. Induced overexpression of a portion (423 bp) of the antisense RNA, spanning the ftsZ AUG codon and the ribosome binding site of ftsZ mRNA, from pBS(KS) could downregulate the synthesis of FtsZ protein to approximately 30%, leading to cell division arrest and filamentation of the cells at 42°C. This effect was less dramatic at 30ºC, probably due to less melting of the antisense RNA. Immunostaining performed on the induced culture did not show FtsZ ring formation after overnight induction whereas reduction in the proportion of the cells carrying FtsZ rings could be clearly observed after 2 hrs of induction. Real time PCR analysis performed for relative quantitation of ftsZ mRNA and ftsZas RNA from different growth phases (0.2 to 2.5 OD600 nm) showed growth phase dependent expression of the antisense RNA. While the levels of ftsZas RNA were found to be high at lower OD cultures or early growth phase cultures, the levels were found to be low at the late log phase and stationary phase cultures. Thus, when the cells are actively dividing and therefore need more FtsZ, the levels of the ftsZas RNA are high, while the cells are not actively dividing and therefore the FtsZ levels are low, the levels of the ftsZas RNA are low. At any phase of the growth, the ratio of the ftsZ mRNA to the ftsZas RNA was always found to be 6:1. Thus, the physiological role the ftsZas RNA is to maintain the availability of the ftsZ mRNA at a level that is commensurate with the requirement for the FtsZ protein during the different stages of the cell growth and division. The Chapter 4 is on the study of the possible mechanism behind the influence of FtsH protease on the presence of FtsZ rings at the mid-cell site during septation in cell division. Immunostaining for FtsZ in the mid-log phase E. coli cells showed that 82% of the AR3289 (ftsH wild type) cells possessed FtsZ rings, while only 18% of the AR3291 (ftsH-null maintained viable by a suppressor mutation) cells showed Z-rings. While the AR3289 cells showed a cell doubling time of 20 min, the AR3291 cells had a cell doubling time of 45 min. The mass doubling time of AR3289 and AR3291 were 24 min and 54 min, respectively. These distinct differences were found in spite of the suppressor mutation suppressing all the deleterious effects of the lack of the essential protease, FtsH. Complementation of the ftsH-null cells (AR3291) with the wild type FtsH but not with the ATP-binding or ATPase, or protease-defective mutants of FtsH, restored the FtsZ ring status to about 80% of the cells. The growth rate of AR3291 was also partly restored to comparable to that of the wild type cells upon complementation. Western blotting for FtsZ, and the FtsZ-stabilising proteins, FtsA and ZipA, showed that the ftsH-null cells have low levels of FtsA, as compared to those in the isogenic wild type cells (AR3289). The levels of FtsZ and ZipA were comparable in both the cells. Quantitative PCR performed for different cell division genes within the dcw cluster showed no sign of change in the ftsA transcript levels in the ftsH-null cells, suggesting that the low levels of FtsA in the ftsH-null cells were not due to transcriptional downregulation. Further experiments showed that the half-life of FtsA protein in the AR3289 cells was 45 min, while that in the AR3291 cells was 24 min. This experiment showed that the low levels of FtsA in the ftsH-null cells was due to the low half-life of FtsA in the cells. Growth synchronisation of the AR3289 and AR3291 cells showed that the levels of FtsA prior to cell division stage do not increase in the ftsH-null cells as much as in the isogenic wild type cells. Thus, the ftsH-null cells must be somehow managing the division through the partial stabilisation of FtsZ rings by ZipA. Interestingly, immunostaining for FtsH in AR3289 cells showed the presence of FtsH at the mid-cell site, as co-localised with FtsZ, for a brief period prior to cell constriction. These observations suggest the involvement of FtsH in cell division process. The faster degradation of FtsA in the absence of FtsH protease implies that another protein, which may be a protease that directly degrades FtsA or a chaperone that helps the unfolding of FtsA for degradation, might be the substrate of FtsH protease. The absence of FtsH protease brings up the levels of this unknown protein, which in turn facilitates (if it is a chaperone) degradation of or directly degrades (if it is a protease) FtsA. This model for the link among FtsH, FtsA levels, and the presence of FtsZ has been proposed based on the observations. Thus, the present study reveals for the first time an FtsA-linked role for FtsH protease in the presence of FtsZ ring at the mid-cell site and hence in bacterial septal biogenesis. The thesis is concluded with the list of salient findings, publications, and references.