Cell differentiation in Caulobacter
The developmental program by which a single cell proceeds to a fully-developed organism involves accurate chromosome replications accompanied by cell divisions that Lucille Shapiro yield dissimilar daughter cells. At each step in the program the characteristics that differenThe generation of cellular asymmetry is the earliest step in a developmental progrartt tiate one daughter cell from the This occurs at each Caulobacter cell division when structural and functional polarity other generally result from difis established in the predit~'ional cell. To do this, genetic mechanisms act to measure ferent temporal programs of time and to organize cell constituents in t h r e e - d i ~ l space. gene expression and, in some cases, from different subcellular positioning of both RNA and protein gene products. How this is brought confined to the predivisional cell and the flagellumabout remains one of the most fundamental questions bearing swarmer daughter cell (Fig. 1). Cell division yields a swarmer cell and a stalked of developmental biology. How is the organization of a single cell changed so that the resulting daughter cell. The swarmer cell is unable to initiate the replicacells differ from one another as well as from the tion of its chromosome until a third of the cell cycle has passed and it has shed its flagellum and begun mother cell? To approach this question, several labs are study- stalk formation2. The stalked cell resulting from cell ing a unicellular bacterium, Cau/obacter crescentus, division initiates DNA replication and behaves like a whose simple life cycle is focused on the generation stem cell, continually forming a 'swarmer' cell pole of asymmetry in the predivisional cell (reviewed in that results in a 'new' swarmer cell and an 'old' Ref. 1). Cell division in this organism always yields stalked cell upon binary fission. Cell division thus daughter cells that differ structurally and func- produces two cells that have different programs of tionally. To understand the mechanisms that estab- temporally-regulated gene expression and DNA lish polarity in the predivisional cell and different pro- replication. grams of gene expression in the daughter cells, it is necessary to define subcellular structure and to iden- The s t r u c t u r e of the flagellum tiff/the genes that are differentially expressed and The localized biogenesis of the flagellum is one of those whose products are positioned at specific poles the major differentiation events in the C. crescentus of the cell. The relatively simple set of changes that cell cycle. It is possible to study the regulatory accompany the manifestation of polarity in C mechanisms that control the synthesis of its comcrescentus and the ability to dissect these changes ponent proteins, the localization of these proteins to both genetically and biochemically has provided a one cell pole, and their assembly into a functional window to the mechanisms controlling temporal structure. To identify and isolate the complete set of gene expression and spatial distribution in a single genes that encode the flagellar structural and cell. regulatory proteins, it is necessary to understand the three-dimensional structure of the flagellum and to be able to assign specific proteins to structural The cell c y c l e The morphological transitions that occur during components. The C crescentus flagellum (Fig. 2) is composed of each C. crescentus cell cycle are shown schematically in Fig. 1. The motile swarmer cell has a single polar basal body, hook and filament subassemblies s. The flagellum and several polar pill. After approximately basal body spans the cell envelope and serves both to one-third of the cell cycle, the flagellum is released anchor the flagellum in the cell and as a rotor. The from the cell and the pili disappear. A polar ring of basal body is composed of five rings that are threaded membrane and cell wall synthesis is then initiated, on a rod 6. The layers of the cell envelope, including resulting in the formation of a tube-like stalk at the the outer membrane, the peptidoglycan, and the site previously occupied by the flagellum. Chromo- inner membrane, are in contact with some of these some replication is also initiated in the new stalked rings. The innermost ring (the M-ring) has a complex cell2. As the stalked cell enlarges and equatorial structure with a button at its centreL There are five constriction begins the replication of the major proteins in the basal body in addition to the 32 chromosome is completed and a new flagellum and kDa rod protein and several minor proteins. Electron micrographs of the puritied hook subseveral pili are assembled at the pole opposite the stalk forming the predivisional cell. The localization assembly were used to derive three-dimensional of flagellar and pill proteins to one cell pole is an reconstructions 7. The hook was found to be comaccessible feature of the polarity established in the posed of protein monomers arranged in a rightpredivisional cell. Another is the polar distribution of handed helix with intersecting families of 5-, 6- and the proteins involved in chemosensory trans- 11-start helices. The sixteen 6-start grooves obserduction 3'4. These are the integral membrane methyl- ved in the hook yield a structure consisting of 295 ± accepting chemotaxis proteins (MCPs), the methyl- 13 elongated 70 kDa monomers. The cell-proximal transferase, which methylates the MCPs, and the portion of the hook is attached to the rod of the basal methylesterase, which cleaves the carboxylmethyl body and the cell-distal portion of the hook joins the ester. The activities of all three types of protein are flagellum filament, itself a right-handed helixs. © 19K5. ~
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Fig. 1. The Caulobacter crescentus cell division cycle showing the time of synthesis (open blocks) of pilirZ"5, flagellar proteins 14 26 and chemotaxis proteins.4 The time of appearance of DNA phage receptoractivity (PRA) is also indicatea¢'Z2R . The activity of several proteins involved in chemotaxis methy lationfunction 3, as a percent of that observed in the swarmer cell, is shoum above the aplrropriate cell stage in the cell cyclediagram. Reprinted, with permission, from Ref. 1. Reprinted, with permission, from Ref. 20. the methylesterase. The expression of all these proteins has been shown to occur at a specific time in the cell cycle (Fig. 1). The flagellins and the hook protein components of the flagellum are synthesized just prior to their assembly in the predivisional celP 4'~5. The synthesis of hook protein and flagellin B is completed prior to cell division but flageUin A synthesis continues in the daughter swarmer cell 14J5.The position of a given protein component within the flagellar structure (the hook protein being closer to the cell than the flagellin A which is assembled at the most distant portion of the filament) may reflect the temporal order of their synthesis. The three identified components of the chemosensory system, the MCP, methyltransferase and methylesterase, are synthesized at the same time as the hook protein and flagellin B (Ref. 4). To examine the molecular signals that control the Temporal regulation of flagellar and temporal expression of these proteins, the genes enchemosensory gene expression The biogenesis of the C. crescentus flagellum ceding several of the flagellar ~6-~a and chemotaxis ~ requires the expression of 27 flagellar (f/a) genes, the proteins have been identified and isolated (Fig. 3). rotation of the flagellum requires three motility (mot) Cloned flagellin and hook protein genes have been genes, and the chemosensory response (che) requires used as probes to demonstrate that the mRNA at least 8 genes (Fig. 3) 12'1s. For these 38 genes, encoding these proteins is regulated as a function of proteins of known function have been assigned to the cell cycle~9-2k Flagellar genes whose products have not yet been only seven loci. These include the hook protein, the three flagellins, an MCP, the methyltransferase and identified were accessed by the insertion of trans-
The flagellar filament is composed of two flagellin monomers of 27.5 kDa (FlaB) and 25 kDa (FlaA). These proteins exhibit immunological crossreactivity and similar amino acid compositions9'1°. Studies of comparable peptide sequences from these proteins showed that they arose from the duplication of an ancestral gene 9.t°. Analysis of flagellar mutants showed that both flagellins are required to assemble a functional filament nJ~. In addition to the hookproximal flagellin B and the distal flagellin A (the major portion of the filament), a 29 kDa flagellin is required for correct filament assembly. The minor 29 kDa flagellin is detected intracellularly in wild-type cells and assembled into short filaments in some motility mutants t2.
" F I G - December 1985
poson-carried, promoter-less, drug resistance genes. The transposon Tn5 was altered so that on insertion into the C. cresczmtuschromosome,fla, che or mot gene transcription fusions are formed. Non-motile mutants that were able to grow in the presence of kanamycin were generated by insertion of the Tn5 element Tn5-VB32 (Ref. 22) carrying a promoter-less neomycin phosphotransferase II (NPT II) gene whose product confers resistance to kanamycin. In these interrupted genes, the promoter regions of the C crescentus flagellar genes drive the expression of the downstream promoter-less NPT II gene. Fusion proteins cannot be formed because of the presence of translation stop codons in all three reading frames 5' to the start of the NPT II gene. Using anti-NPT II antibody, the synthesis of NPT II was measured in synchronized cultures of non-motile insertion mutants that had been pulse labeled with ]4C-amino acid at different stages of the cell cycle. The synthesis of NPT II was found to occur in these mutants at the same stages in the cell cycle as synthesis of the wild-type flagellar and chemosensory $ene products (Champer and Shapiro, unpublished~. Therefore, the 5' regulatory regions of insertionaUy-inactivated flagellar genes were found to control the temporal expression of NPT II. The temporal control off/a and che gene transcripts occurs either by the modulation of mRNA initiation, by mechanisms analogous to attenuation, or by changes in mRNA stability.
The e x p r e s s i o n of flagellar and c h e m o t a x i s genes is regulated by a cascade of transacting factors Accumulating evidence suggests that the regulatory events controlling the time at whichfla and che genes are expressed, the positioning of their gene products, and the ordering of the assembly process are not mutually exclusive. Several groups of f/a and che genes, whose temporal expression is coordinated, are widely scattered in the C. crescentus genome. These genes could be 'turned-on' by an activation factor that recognizes a common sequence within the 5' regulatory regions, or they could be 'turned-on' serially in a regulatory cascade in which the product of one gene turns on the expression of a second set of genes, and so on. Two sets of experiments have provided evidence that f/a and che gene expression is controlled by a transacting regulatory cascade. The gene f/a Y lies 3' to the gene encoding the 29 kDa flagellin. Tn5 insertions in f/a Y and a temperature-sensitive mutant inf/a Yare non-motile and synthesize very little of the three flagellins and the chemotaxis MCPs, methyitransferase, and methylesterase, all of which map elsewhere on the chromosome (Fig. 3)23. Complementation by an intact f/a Y gene carried on a low-copy-number plasmid restored normal synthesis of all the flagellin and chemotaxis gene products23. Thus, the product ofthef/a Ygene in trans controls the level of synthesis of the 3' adjacent flagellin genes and the more distal chemotaxis genes. Thef/a Y gene product does not control hook protein synthesis, but the expression of aria gene that maps 3' to the hook protein structural gene is required for the synthesis of normal amounts of the flagellins and the hook protein z4. These results suggest that a
filament I " [email protected]
: 25k FlaA 27.5k FlaB [29k] FlaB + FlaA FlaB
basal bod~ 32k- rod ~Oek
89k I - 5 rings 81k 52k 45
Fig. 2. The C. crescentus flagellum. The flagellar pro~'n cornOonents are indicated.
common activation factor is not solely responsible for the 'turn-on' of the co-ordinately expressed f/a and che genes. Additional evidence for a tram-acting regulatory hierarchy comes from experiments in which flagellar gene fusions with the promoter-less NPT II gene were transduced into strains carrying point mutations or deletions in specific flageUar genes. The purpose of creating these double mutants was to determine if a mutation in a given flagellar gene would trsnsf~ase
29 kDa [email protected]
Fig. 3. Thef14~l~ (~), d~ao~.:s (che)and ~ (mot) genes of Caulobacter crescentus CB15. The map po~'tions of these genes were determined by Ely and co-mTrkerszs. Cloned genes are enclosed in boxes. Data taken from Refs. 16-18.
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Spatial organization and the generation of polarity The synthesis and assembly of a flagellum in the predivisional cell requires that a group of newly synflaYp ,mRNA thesized f/a proteins be positioned at one cell pole. Upon division, the flagellum is partitioned to the , )( flaS ~IrNPT II Tet daughter swarmer cell. After division has taken r~ genome ! I place, the swarmer cell flagellar filament continues naVp ) ( , mRNA to elongate. The proteins involved in chemosensory transduction are also known to be present in the pre(b) Hierarchy of control divisional cell and the daughter swarmer celP, yet f/aS they are not part of the flagellar structure. What are the mechanisms used by the predivisional ceU to position the fla and cke proteins so that they are asymmetrically distributed on cell division? In the case of the chemosensory MCPs, it has been ¢ ¢ demonstrated that those proteins are synthesized in flagellins chemotaxis proteins the predivisional cell only, along with the hook protein and flagellin B (Ref. 4). Like these proteins, the Fig. 4. The use of double mutants to detect the effect of trans- MCPs are not synthesized in either of the two acting factors on the expression of transcription fusions between daughter cells, yet the proteins can be detected fla gem regulatory regions and an adjacent promoter-less drug immunologicaUy and function solely in the daughter resistance gene (NPT II). (a) A representation of the flaSgenepro- swarmer cell. MCPs pulse-labeled in the preduct functioning in trans to control the level of the flaY-NPT H divisional cell can be chased, specifically, into the transcript. If the flaS gene product/s not synthes/zed, the transcript encoding N P T H is not available. (b) A hierarchy of trans- swarmer cell upon division4. In addition, two soluble acting control based on ¢omplementation with cloned genes and proteins involved in the chemosensory machinery, the level of N P T H synthesized in double mutants carrying Tn5- the methyltransferase and the methylesterase, are VB32 insertionally-inactivated fla genes. Reprinted, in part, also partitioned to the swarmer cell4. The generation of polarity in the predivisional cell from Ref. 20. involves more than the correct distribution of proteins. For example, Milhausen and Agabian have result in the 'down regulation' of a second flageUar shown that the mRNA for one of the flagellins, Fla A, gene, for example, f/a Y (Fig. 4) whose expression is synthesized in the predivisional cell only but is then can be monitored by the immunological detection of segregated to the daughter swarmer cell19. The NPT II synthesis. Because a protein fusion between flagellar filament of the swarmer cell continues to the product of the gene encoding NPT II and the lengthen with the addition of Fla A monomers that interruptedf/a Ygene cannot occur, any trans-acting result from the translation of the mRNA originally effect must occur at the level of the transcript. The synthesized in the predivisional cell. rationale for this experiment is shown in Fig. 4. To study the mechanism whereby the flageUar and Mutations in flaS were shown to cause a significant chemosensory receptor proteins and, in some cases, decrease in NPT II synthesis (and thus kanamycin their transcripts, are positioned in the cell, the resistance) under the control of the promoter region of segregation of the promoter-less NPT II gene prof/a Yand several otherfla genes 2°. duct, driven by 5' regulatory regions of flagellar The identification of flagellar genes whose pro- genes, was tracked immunologicaUy. We have ducts are required for the synthesis of normal recently found that the promoter-less NPT II drugamounts of flagellin and chemotaxis proteins whose resistance protein is positioned in the same manner genes map elsewhere on the chromosome 2°'2sand for as a flagellar gene product when it is inserted within a the expression of flagellar genes of unknown specific chromosomal flagellar gene. NPT II expresfunction that have been accessed by a promoter-less sion is driven by the 5'-regulatory regions of these NPT II gene 2°, has allowed the construction of the genes (Champer and Shapiro, unpublished). These hierarchy of trans-acting control shown in Fig. 4. results, together with the observed temporal regulaThis hierarchy is based on the observations that the tion of the NPT II gene expression, argue that the f/a Y gene product is required for flagellin and 5'-regulatory regions of thef/a and che genes can be chemotaxis gene expression, and that f/a Y expres- involved not only in the timing of their expression, sion, in turn, is dependent on the product off/aS. This but also in the positioning of their gene products regulatory cascade is analogous to that shown to during the generation of asymmetry. It can now be function in E. coli for the regulation of flagellar and determined if this positioning is controlled by specific chemotaxis gene expressionzS. Komeda has mRNA targeting sequences in the 5'-regulatory suggested that in E. coli, the position of a given gene regions of these genes, or by specific transcription in the regulatory cascade reflects the position of the from only one of the two newly-replicated chromostructural gene product in the flagellum. Thus, the somes in the predivisional cell. innermost, or most cell-proximal, protein components are at the top of the hierarchy and are synthesized first, and the most cell-distal protein com- R e f e r e n c e s ponents are at the bottom of the hierarchy (i.e. flagel1 Shapiro, L. (1985) Generation of polarity during Caulobacter cell differentiation. Annu. Rev. Cell Biol. 1,225-59 lins) and are synthesized and assembled last. ~aS
~' NPT II Tot
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2 Degnan, S. T. and Newton, A. (1972) Chromosome repli- 17 Purucker, M., Bryan, R., Amemiya, K., Ely, B. and Shapiro, L. (1982) Isolation of a Caulobacter gene cluster specitying flagelcation during development in Caulobacter~ t u s . ]. Mot Blot lum productionby using non-motileTn5 insertionmutants. Pr~. 64, 671-80 3 Shaw, P., Comes, S. L., Sweeney, K., Ely, B. and Shapiro, L. Natl Aaut. Sci USA 79, 6797-6801 (1983) Methylation involved in chemotaxis is regulated during 18 Mil_hausen,H., Gil, P. R., Parker, G. and Agabian, N. (1982) Cloning of developmentally-regulated llagellin genes from CauloCaulobacter differentiation. Pn¢_. Natl Acad Sd. USA 80, 5261 4 Comes, S. L. and Shapiro, L. (1984) Differential expression batter crescem~ v/a immunoprecipitation of polyribonomes. ]Woc. Natl Acad, Sci. USA 79, 6847-6851 and ix~sitiortingof chenmtaxis methylation proteins in ~ b a c t e r . 19 Milhausen, M. and Agabian, N. (1983) Cmdobacter flagellin J. Mot Biol 178, 551-568 5 Johnson, R. C., Walsh, J. P., Ely, B. and Shapiro, L. (1979) mRNA segregates asymmetrically at cell division. Nature 302, Flagellar hook and basal complex of Caulobacter crescgntus,f Bac- 630qi32 20 Champer, R., Bryan, R., Comes, S. L., Purucker, M. and ter/ol. 138, 984 6 Stallmeyer, M. J. B., De Rosier, D. J., Aizawa, S.-I., Macnab, Sbaprio, L. Temporal and spacial control of flagellar and chemoR. M., Hahnenberger, K. and Shapiro, L. (1985) Structural Studies taxis gene expression during Caulobactercell differentiation. Co/d Slm'ng Harbor Syrup. Quant BioL (in press) of the basal body of bacterial flagella. Biophys. f , 47-48a 7 Wagenknecht, T., De Rosier, D., Shapiro, L. and Weissborn, 21 Newton, A., Ohta, N., Huguenel, E., Chen. L.-S. (1985} in A. (1981) Three-dimensional reconstruction of the flagellar hook Spores IX (Setlow, P. and Hoch, J., eds.) Am. Soc. Microbiol. (in from Caulobactercrescentus.]. Mot Biol. 151,439 press) 8 Koyasu, S. and Shirakihara, Y. (1984) Caulobactercrescentus 22 Bellofatto, V., Shapiro, L. and Hodgaon, D. (1984) Generallagellar filament has a right-handed helical form.]. Mot BioL 173, fion of a Tn5 promoter probe and its use in the study of gene expression in Caulobaaer crescentus.Proc. NatlAcad Sci. USA 81, 1035 125 23 Bryan, R., Purucker, M., Comes, S. L. Alexander, W. and 9 Gil, P. R. and Agabian, N. (1982) A comparative structural analysis of the flagellin monomers of Caulobacter crescen/us Shapiro, L. (1984) Analysis of the pleiotropic regulation of flagellar indicates that these proteins are encoded by two genes. J. Bacte'n;o/. and chemotaxis gene expression in ~ crescent~ using plasmid complementation. Proc. Natl Acad. Sci USA 81, 1341 150, 925 I0 Weissborn, A., Steinman, H. M. and Shapiro, L. (1982) 24 Ohta, N., Swanson, E., Ely, B. and Newton, A. (1984) PhyCharacterization of the proteins of the Cgulobacter crescentus sical mapping and complementation analysis of Tn5 mutations in flagellar filament: peptide analysis and filament organization. ]. Caulobactercrescentus: organization of transcriptional units in the Blot Chem. 257, 2066 hook cluster, f Bacter/ot 158, 897 11 Fukuda, A., Asada, M., Koyasu, S., Yoshida, H., Yginuma, 25 Komeda, Y. (1982) Fusions of flagellar operons to lactose K. and Okada, Y. (1981) Regulation of polar morphogenesis in genes on a Mu loc bacteriophage. J. Bacter/ot 150, 16 Caulobaaer ~ t u s . J. Bacterlo/. 145, 559 26 Agabian, N., Evinger, M. and Parker, E. (1979) Generation of 12 Johnson, R. C., Ferber, D. M. and Ely, B. (1983) Synthesis asymmetry during development. ]. Cell Blot 81, 123-36 and assembly of flagellar components by Caulobacter crescentus 27 Lagenaur, C., Farmer, S. and Agabian, N. 0974) Absorption properties of stage-specific Caulobacter OCbk. Virology 77, motility mutants. J. Bacter',bl. 154, 1137 13 Ely, B., Croft, R. H. and Gerardot, C. J. (1984) Genetic map- 401-407 ping of genes required for motility in Caulobacter crescentus.Gene- 28 Huguenel, E. D. and Newton, A. (1982) Localization of surface structures during prokaryotic differentiation: role of cell divit ~ 108, 523-532 14 Osley, M. A., Sheffrey, M. and Newton, A. (1977) Regulation sion in Caulobacter ~ t u s differentiation. Lhfferentiation 21, of flagallin synthesis in the cell cycle of Caulo&u'~. dependence on 71-78 DNA replication. Cell 12, 393-400 15 Lagenaur, C. and Agabian, N. (1978) Caulobacter flagellar organeUe: synthesis, compartmentation and assembly.]. Bactenbl. 135, 1062 16 Ohta, N., Chen, L.-S. and Newton, A. (1982) Isolation and L Shapiro is at the Department of Molecular Biology, Division expression of cloned hook protein gene from Caulobacter cres- of Biological Sdences, Albert Eins~'n College of Med~'ne, centus, t~,oc. Natl Acad. Sci. USA 79, 4863-4867 Bronx, N Y 10461, USA.
Genetics and biology of mouse melanocytes: mutation, migration and interaction
In t h e mouse, m e l a n o c y t e s (the p i g m e n t - p r o d u c i n g cells) have an interesting embryological history, originating in t h e neural c r e s t of t h e e m b r y o and s u b s e q u e n t l y m i g r a t i n g to colonize t h e skin. W h e n in t h e hair follicle, m e l a n o c y t e s can lan J. Jackson p r o d u c e both yellow phaeomelanin and black eumelanin, Coat coloration in the mouse provides a convenient assay for new mutations t h e p i g m e n t s w h i c h colour and a model f o r complex g e m interactions. The interesting embryological m o u s e hair. T h e d e r m a l cells of behaviour o f melanocytes (pigment-producing cells) serves as a good model f o r the hair follicle regulate syncell migration during development and the action o f genes on this migration. thesis and deposition of t h e pigGenes closely linked to coat colour genes have beenf o u n d to have speafic effects m e n t s by t h e m e l a n o c y t e s , in development. switching s y n t h e s i s b e t w e e n eumelanin and p h a e o m e l a n i n so that in t h e wild-type m o u s e t h e dorsal hairs have a t h e m o r e interesting aspects, particularly w h e r e black tip and black base s e p a r a t e d b y a yellow band. r e c e n t p r o g r e s s has b e e n m a d e . T h e ventral coat of the wild-type m o u s e a p p e a r s I focus on t h r e e well studied loci. T h e albino (C) lighter b e c a u s e s o m e hairs lack t h e b a n d e d pattern, locus on c h r o m o s o m e 7 has s e v e n well characterized instead h a v i n g simply a yellow tip a n d black base. alleles and m a n y radiation-induced mutations, m o s t of O w i n g to their rich g e n e t i c diversity, t h e coat colour w h i c h s h o w pleiotropic effects on e m b r y o n i c developg e n e s have r e c e i v e d m u c h attention, and I have not m e n t as a result of deletions of n e i g h b o u r i n g genes. a t t e m p t e d a c o m p r e h e n s i v e c o v e r a g e of t h e field (for M u t a t i o n s at C reduce a m o u n t s of both p i g m e n t types. this see Silvers, Ref. 1). Instead, I highlight s o m e of T h e agouti (A) locus on c h r o m o s o m e 2 has over 15 © 1965.ElnevierS,cien¢~~
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