Centrum Algatech

Mikrobiologický ústav AV ČR, v.v.i. - vědecké pracoviště Třeboň

Biologie a fyziologie regulace buněčného cyklu

Vilém Zachleder

Due to more than 40 years of continuous research of the cell cycle of algae the know how obtained is represented by unique and very extensive range of outputs in both basic and applied research. For more details see CV and pages of Milada Vítová and Kateřina Bíšová.

The topics studied recently are: (i) the role of environmental factors like light intensity and temperature or different compounds, (selen, rare earth elements) on the cell cycle progression; (ii) study of the molecular mechanisms responsible for regulation of algal cell cycle, progression into mitosis and response to DNA damage; (iii) development of processes allowing usage of waste CO2 and waste waters for algal biomass production, with controlled synthesis of large amounts of starch and lipids for a various applications including bioethanol and biofuels production.

 

Outputs of basic research in 2006-2011

 

 

 

Diagrams showing the types of the cell cycle phases and their ordering in a classical cell cycle model and those found in the algae Scenedesmus andChlamydomonas dividing into two (A) and four (B) daughter cells.

Classical cell cycle model after Howard and Pelc (1953), Scenedesmus cell cycle model after Šetlík and Zachleder (1984), Chlamydomonas cell cycle model after Zachleder and van den Ende (1992). Individual stripes indicate the sequence of cell cycle phases during which growth and reproductive processes leading to duplication of reproductive structures take place. Whereas only one sequence of events leading to duplication of cell structures occurs during the cell cycle of cells dividing into two daughter cells (panel A), two, partially overlapped sequences of growth and reproductive events occur within one cycle in cell dividing into four daughter cells (panel B). Two stripes (panel B) illustrates simultaneous course of different phases from two consecutively started sequences of growth and reproductive events. Schematic pictures of cells indicate their size changes during the cell cycle and the black spots inside illustrate the size and number of nuclei. Large black spots indicate doubling of DNA. G- the phase during which the threshold size of the cell is attained. It can be called a precommitment period because it is terminated by attainment of the commitment point. C.P. - the state of the cell cycle at which the cell becomes committed to triggering and terminating the sequence of processes leading to the duplication of reproductive structures. pS - the prereplication phase between the commitment point and the start of the round of DNA replication. The processes required for the initiation of DNA replication are assumed to happen during this phase. S ‑ the phase during which DNA replication takes place. G2 ‑ the phase between the termination of DNA replication and the start of mitosis. Processes leading to the initiation of mitosis are assumed to take place. M - the phase during which nuclear division occurs. G3 ‑ the phase between nuclear division and cell division. The processes leading to cellular division are assumed to take place during this phase. C ‑ the phase during which the cleavage of cells occurs.

 

Grants

1. MŠMT ME 104 (2006-2007): Study of the answer reparation-defect strains of the alga Chlamydomonas reinhardtiito DNA damage in relation to regulation of cell cycle. Principal investigator Zachleder, V.

2. GAČR 204/06/0102 (2006-2008): Regulation of mitosis by plant specific cyclin-dependent kinasis type B in green alga Chlamydomonas reinhardtii. Principal investigator Bišová, K.

3. GAAV A500200614 (2006-2010): Regulation of mitosis by kinase WEE1 using the green alga Chlamydomonas reinhardtii as a model organismsPrincipal investigator Bišová, K.

4. GAAV A600200701 (2007-2009): Výzkum akumulace selenu a jeho forem v průběhu buněčného cyklu zelených řas. Principal investigator Vítová, M.

5. GAČR 204/09/0111 (2009-2011): Příprava teplotně sensitivních mutantů buněčného cykluu zelené řasy Chlamydomonas reinhardtii. Principal investigator Bišová, K.

6. GAČR 525/09/0102 (2009-2011): Bioaccumulation of rare earth lements by green algae, effect on physiology anf cell cycle. Principal investigator Vítová, M.

7. GAAV M200200904 (2009-2012): Role of endogeneous timers in regulationships between growth and cell division of algae. Principal investigator Zachleder, V.

8. GAČR P501/10/P258 (2010-2012): Role of phosphatases in regulation of CDK in G2/M phase of cell cycle Chlamydomonas reinhardtii. Principal investigator Čížková, M.

 

Outputs of applied research in 2006-2011

 

 

Changes in biomass and starch content (A) and starch yield per biomass (B) in cultures of Chlorella. The cultures were grown in an outdoor scale up thin layer photobioreactor in complete mineral medium for 120 h and then in sulphur limiting medium (–S). Dark periods (nights) are marked by black stripes and separated by vertical solid lines

 

Changes in biomass and starch content (A) and
starch yield per biomass (B) in cultures of Chlorella.
The cultures were grown in an outdoor scale up
thin layer photobioreactor in complete mineral
medium for 120 h and then in sulphur limiting
medium (–S). Dark periods (nights) are marked
by black stripes and separated by vertical solid lines
Fluorescence microphotographs of Parachlorella kessleri treated
to produce increased amount of lipid reserves
Red color: autofluorescence of chlorophyll
Yellow collor: vesicules of lipid reserves
Electron microscopic photomicrographs
of daughter (A) and mother cells (B) of
the alga Chlorellagrown in complete
nutrient medium (A,B) and the cells
grown in the presence of cycloheximide
(1 mg/L) (C) and in nutrient medium,
where sulfates were omitted (D).
Nu nucleus, St starch granules, Bars = 20 μm

Methods

1. Synchronization of algal cultures

Zachleder V., Šetlík I.(1990): Timing of events in overlapping cell reproductive sequences and their mutual interactions in the alga Scenedesmus quadricauda. Journal of Cell Science 97: 631-638.

2. Characterization of cell cycle progress:

2a Analysis of RNA and DNA content and timing of DNA replication

Zachleder V.(1986): Optimization of nucleic acids assay in green and blue-green algae: Extraction procedures and the light-activated reaction for DNA. Arch. Hydrobiol. (Suppl. 67) Algolog. Stud. 36: 313-328.

 

2b Assessment of commitment curves

Zachleder V., Schläfli O., Boschetti A. (1997): Growth-controlled oscillation in activity of histone H1 kinase during the cell cycle of Chlamydomonas reinhardtii(Chlorophyta). Journal of Phycology 33: 673-681.

Vítová M.Zachleder V. (2005): Points of commitment to reproductive events as a tool for analysis of the cell cycle in synchronous cultures of algae. Folia Microbiologica: in press.

2c Determination of number of nuclei by means of DNA staining by SYBR Green I or DAPI, nuclear division curves

Zachleder V.(1986): Nuclei stained by SYBR Green I (left) and by DAPI (right) in Scenedesmus quadricauda

Vítová M., Hendrychová J., Cepák V., Zachleder V.(2005): Visualization of DNA containing structures in Chlamydomonas reinhardtii and other species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye. Folia Microbiologica: in press.

Zachleder V., Cepák V.(1987): Visualization of DNA containing structures by fluorochrome DAPI in those algal cells which are not freely permeable to the dye. Arch. Hydrobiol. (Suppl. 78) Algolog. Stud. 47: 157-168.

 

3. Cyclin-dependent kinase activity assay in vitro

Bišová K.Vítová M.Zachleder V.(2000): The activity of total histone H1 kinases is related to growth and commitment points while the p13(suc1)-bound kinase activity relates to mitoses in the alga Scenedesmus quadricauda. Plant physiology and biochemistry 38: 755-764.

4. Single CDK complex activity assay in-gel

5. Nuclear transformation of Chlamydomonas using glass beads

Pacanovská M.(2003): Construction of plasmid with E.coli recA+ gene to increase frequention of homologous recombination in Chlamydomonas reinhardtii. MSc. thesis. Comenius University, the Faculty of Natural Sciences, Bratislava, Slovak Republic.

Slaninová M., Fritsche L., Pacanovská M., Fuseková M., Treuner G., Vlček D., Mages W.(2004): New approaches to improve homologous recombination in Chlamydomonas reinhardtii. In book of European Environmental Mutagen Society (Maastricht, the Netherlands).

6. Fluorescence microscopy (Olympus fluorescent microscope BX51 with attached CCD camera, Nomarski (DIC) and phase contrast)

Vítová M., Hendrychová J., Cepák V., Zachleder V.(2005): Visualization of DNA containing structures in Chlamydomonas reinhardtii and other species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye. Folia Microbiologica: in press.

Hendrychová J., Vítová M.Bišová K., Wiche G., Zachleder V.(2002): Plectin-like proteins are present in cells of Chlamydomonas eugametos (Volvocales). Folia Microbiologica 47: 535-539.