Diferenciace heterocyst Martin Tichý Martin Tichý Institute of Microbiology, Czech Academy of Sciences, Třeboň Institute of Physical Biology, University of South Bohemia, Nové Hrady Czech Republic

Anabaena sp. PCC 7120 je Nostoc heterocysty nejsou cysty

Paradox of developmental biology How is it that a single cell gives rise to a multicellular organism composed of 100s of different cell types – yet all the cell types have the same genes?

Patterning can involve the interpretation of positional information. French flag analogy

Figure Two strategies for using signal concentration gradients to specify a fine-grained pattern of cells in different states. In (A) there is only one signal gradient, and cells select their states by responding accurately to small changes of signal concentration. In (B) the initial signal gradient controls establishment of a small number of more local signals, which control establishment of other still more narrowly local signals, and so on. Because there are multiple local signals, the cells do not have to respond very precisely to any single signal in order to create the correct spatial array of cell states. Case B corresponds more closely to the strategy of the real embryo.

Expression of eve Stripe 2

Figure The formation of ftz and eve stripes in the Drosophila blastoderm. Genes ftz and eve are both pair-rule genes. Their expression patterns (shown in brown for ftz and in gray for eve) are at first blurred but rapidly resolve into sharply defined stripes. (From P.A. Lawrence, The Making of a Fly. Oxford, UK: Blackwell, 1992.)

The Course of Development

Time Events in time and space... Complicated

The Course of Development TimeComplicatedReally

heterocysts Matveyev and Elhai (unpublished) N2N2 NH 3 Cyanobacteria Anabaena grown without fixed nitrogen

Paradox of developmental biology How is it that a single cell gives rise to a multicellular organism composed of 100s of different cell types – yet all the cell types have the same genes? How Cyanobacteria Count to 10 Robert Haselkorn Jak každá desátá buňka ví že má být heterocystou

Fixace dusíku

N 2 + 8H + + 8e ATP --> 2NH 3 + H ADP + 16Pi Note: Very expensive Reason why N 2 fixation by heterotrophic microbes is probably low Key enzyme: nitrogenase (nif) Ancient enzyme: highly conserved in very diverse microbes, from archaea to cyanobacteria Biochemistry of N 2 fixation

What is another problem with nitrogenase ? Nitrogenase is killed dead by O 2 Protects nitrogenase (N 2 fixing enzyme) from O 2 Outside sources of O 2 O 2 produced by cyanobacteria

Ne všechny sinice schopné fixovat dusík tvoří heterocysty

How does Trichodesmium (and single cell cyanos) fix N 2 without heterocysts? Partial answer: doesnt fix N 2 and do photosynthesis at the same time See Berman-Frank et al. Science (2001) 294:

1. Site of N 2 fixation in many cyanobacteria. 2. Specialized thick wall cells in chain of cyanobacterial vegetative cells 3. No PS II of photosynthesis --> no O 2 evolution 4. No carbon fixation 5. Respiration What are heterocysts?

The heterocyst achieves a near anoxic state by at least three means. First, photosystem II, the O 2 -producing end of the photosynthetic electron transport chain, is dismantled during heterocyst differentiation, so that the heterocyst need contend only against O 2 produced by neighboring vegetative cells and that dissolved in the environment. Second, heterocysts are invested with a specialized envelope that limits the influx of gases. Two layers within the envelope have been implicated in O 2 protection: an inner layer composed of a hydroxylated glycolipid and an outer layer of polysaccharide. Neither layer is found in vegetative cells. Third, much of the O 2 that overcomes these barriers is consumed by the high oxidase activity associated with heterocysts.

Dělba práce

Excitation was at 510 to 560 nm (green), exciting phycoerythrin, and emission was greater than 600 nm. Heterocysts have negligible fluorescence, while vegetative cells have intense combined fluorescence from phycobiliproteins and chlorophyll a. Bar, 10 µm. A médium s dusíkem B médium bez dusíku

Anabaena filament growing on nitrate Anabaena filament growing on N 2 removal of nitrate 18 hours Heterocysts only when needed

Anabaena heterocyst cells vegetative cells

Anabaena model Heterocyst spacing relatively constant –Heterocyst cells produce compound –Vegetative cells divide differentiate consume compound diffuse compound

First, they assumed that any cell is competent to differentiate at the moment when nitrogen is removed from the environment and that the choice of cells that initiate differentiation is random. Second, they postulated the existence of a diffusible inhibitor made by heterocysts and differentiating cells and consumed by nondifferentiating cells, as predicted by experimental data.

Anabaena – continuous model axiom: F h (s max,c max ) F v (s max,c max ) F h (s max,c max ) F(s l,c l ) F(s r,c r ): if s c min solvedc/dt = D. (c l +c r -2c)-µ. c ds/dt = r. s if s = s max & c > c min produce F v (k. s max,c)F v ((1-k). s max,c) if c = c min produce F h (s,c) F h (s,c): solveds/dt = r s. (s max -s) dc/dt = r c. (c max -c)

Anabaena – continuous model

Case of the Hidden Heterocyst heterocysts Matveyev and Elhai (unpublished) N2N2 NH 3 O2O2

Case of the Hidden Heterocyst Strategy to find heterocyst differentiation genes Nostoc genome Transposon 1. Use transposon mutagenesis

Case of the Hidden Heterocyst Strategy to find heterocyst differentiation genes Nostoc genome Transposon 1. Use transposon mutagenesis to find a mutant defective in heterocyst differentiation

Case of the Hidden Heterocyst Strategy to find heterocyst differentiation genes Nostoc genome 2. Sequence out from transposon AAGCTTGACCAAAAAGTTAAAACACTGACGGCAAATA ATCAATGACTATCAGACAGAGAATCATCGTGCTGTCA GTAAAACCTCTGATTTCGATCTTTACCATAATTGTTA TGTTGTAATGACTAACCAGACTATCTTTTACAGAGCT TCTGGTTAACACTTGTCTAATTAGACATTGATAATGT TTGTGGGGGTTGGTCATCAGGAATGGTAAATAGCAAT TACCCTTCAGACTTTCCTATGAGACGCTCCGCCAACG AGCAGTGTCTCTTAAAGAACGTTATGAGCGCTCAGTT AACTTCAGAAATTCACGGCGGAAATCCATAGTTATTA TTACTTATGACTAAAACAAAATTACTATGGCGGCTTG TTTAATATAGATTCTGTGTTCTGAGAAATGACTTTTA AAGTCCCACTAACTTTTTTCTCATCTATTGCTATATT TCGACTTTAAAACTTATAGTAGATGGCTTAATTCTCA AATAACAAACTCATTTTTAGTAGATATTTCATGCAAA CTGAGGTTTTTAGTGATATTTTCCCCTTATTGAGTAC AGCCACTCCACAAACCTTAGAATGGCTACTCAATATT GCAATTGATCATGAATATCCCACTGGTAGAGCAGTTT TAATGGAAGATGCCTGGGGTAATGCAGTTTATTTCGT TGTATCTGGATGGGTAAAAGTTCGGCGCACCTGTGGA 1. Use transposon mutagenesis to find a mutant defective in heterocyst differentiation

Case of the Hidden Heterocyst Strategy to find heterocyst differentiation genes Nostoc genome 2. Sequence out from transposon AAGCTTGACCAAAAAGTTAAAACACTGACGGCAAATA ATCAATGACTATCAGACAGAGAATCATCGTGCTGTCA GTAAAACCTCTGATTTCGATCTTTACCATAATTGTTA TGTTGTAATGACTAACCAGACTATCTTTTACAGAGCT TCTGGTTAACACTTGTCTAATTAGACATTGATAATGT TTGTGGGGGTTGGTCATCAGGAATGGTAAATAGCAAT TACCCTTCAGACTTTCCTATGAGACGCTCCGCCAACG AGCAGTGTCTCTTAAAGAACGTTATGAGCGCTCAGTT AACTTCAGAAATTCACGGCGGAAATCCATAGTTATTA TTACTTATGACTAAAACAAAATTACTATGGCGGCTTG TTTAATATAGATTCTGTGTTCTGAGAAATGACTTTTA AAGTCCCACTAACTTTTTTCTCATCTATTGCTATATT TCGACTTTAAAACTTATAGTAGATGGCTTAATTCTCA AATAACAAACTCATTTTTAGTAGATATTTCATGCAAA CTGAGGTTTTTAGTGATATTTTCCCCTTATTGAGTAC AGCCACTCCACAAACCTTAGAATGGCTACTCAATATT GCAATTGATCATGAATATCCCACTGGTAGAGCAGTTT TAATGGAAGATGCCTGGGGTAATGCAGTTTATTTCGT TGTATCTGGATGGGTAAAAGTTCGGCGCACCTGTGGA 1. Use transposon mutagenesis to find a mutant defective in heterocyst differentiation 3. Find gene boundaries 4. Identify gene Do it

HetR mutant - unable to make heterocysts The spatially patterned differentiation of heterocysts in the filamentous cyanobacterium Anabaena requires a functional hetR gene low level of transcript when Anabaena is grown with combined nitrogen induction begins within 2 h following nitrogen deprivation by 3.5 h, induction is localized to spaced foci by 6 h, 20-fold increase within spatially separated cells positive autoregulation

HetR Genes needed for differentiation Master regulator Differentiation in cyanobacteria Integration of signals through HetR Position in filament Position in cell cycle Nitrogen deprivation ?? ?? ??

Promoter NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetR gene How might hetR be controlled? 5-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN 3-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN RNA Polymerase Presence of fixed nitrogen Regulatory protein No HetR protein Transcription Absence of fixed nitrogen

NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetR gene 5-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN 3-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN RNA Polymerase Absence of fixed nitrogen How might hetR be controlled?

NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetR gene 5-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN 3-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN RNA Polymerase Absence of fixed nitrogen How might hetR be controlled? HetR protein Transcription

The key result of this experiment is that all of the upstream controls of HetR expression can be bypassed; expression of HetR alone suffices to turn on the differentiation pathway. HetR overexpression

PatS overexpression of patS completely blocks heterocyst development patS encode a 17- or 13-amino-acid peptide, is crucial for the formation and maintenance of the normal heterocyst pattern

Wild-type filaments (A) grown in BG-11 medium and (B) after the nitrogen step-down in BG to induce heterocysts (arrowheads) are shown. (C) Overexpression of patS prevented heterocyst formation in BG-11 0, and (D) deletion of patS resulted in supernumerary heterocysts with an abnormal pattern in BG Differential interference contrast micrographs were taken before (A) and 24 hours after (B through D) heterocyst induction. patS controls heterocyst development in Anabaena PCC 7120

The exogenous addition of a pentapeptide corresponding to the last five COOH-terminal residues of PatS also inhibited heterocyst differentiation, indicating that a processed form of PatS may be a diffusible inhibitory signal regulating development.

Přesně jako v modelu z roku 1975 The inhibition of neighboring cells by select differentiating cells (lateral inhibition) is an important mechanism of pattern formation in eukaryotic organisms.

Because it takes ~20 hours for heterocysts to mature and begin supplying fixed nitrogen to the filament, a specialized early inhibitory signal is required to allow only a fraction of starving cells to terminally differentiate. The first cells to differentiate increase the production of PatS to inhibit neighboring cells from forming heterocysts. PatS- producing cells must themselves be refractory to the PatS signal.

+ Nostoc punctiformeAnthoceros punctatus.

Další události nezbytné k aktivaci nif genů