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Structural proteins and their complexes

This research group is part of Structural Biology and Protein Engineering research program

Research Focus

Structural proteins that form cytoskeletal networks form the internal dynamic scaffold of living cells. These networks enable cells to generate mechanical forces, providing their rigidity and driving adaptive changes in their shape and three-dimensional organization. Thus, the cytoskeleton is essential for key cellular processes, such as cell division, cell motility or morphogenesis. How the individual structural elements of the cytoskeleton mechanically work together to produce coherent behavior of the cytoskeletal network during these processes is not understood.

We address these questions using two main strategies, a bottom-up approach – reconstituting elements of the cytoskeletal networks from individual components in vitro and a top-down approach – deconstructing the function of these networks in vivo. We use genetic manipulations, biochemical and biophysical methods including imaging and manipulation of single molecules by a combination of microscopy and optical tweezers. To get quantitative insight into the functioning of the networks we accompany our experimental observations with mathematical modeling.

Our research is led by two independent scientists, Zdenek Lansky and Marcus Braun.

We are always looking for talented students and postdocs, experimentalists or theoreticians, with a degree in (bio)physics, chemistry, biology or an equivalent field, motivated to work on cross-disciplinary projects. To apply, please contact Zdenek Lansky (zdenek.lansky@ibt.cas.cz) or Marcus Braun (marcus.braun@ibt.cas.cz).

Selected Papers:

Lansky Z et al, Diffusible crosslinkers generate directed forces in microtubule networks, Cell 2015, 160(6): 1159-68

Braun M et al, Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart, Nat Cell Biol 2011, 13(10): 1259-64

Braun M et al, The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism. Nat Cell Biol 2009, 11(6):724-30

Verbrugge S et al, Kinesin’s step dissected with single motor FRET. Proc Natl Acad Sci USA 2009, 106(42): 17741-6

www_video1_single_Ase1_diffusion-2_edited Single microtubule crosslinkers Ase1 tagged with GFP (green) diffuse along microtubules (red). Video was acquired using TIRF microscopy.
www_video2_MT_MT_diffusion_cyan_red-2_edited Microtubules cross-linked by Ase1 diffuse along each other. Long biotinilated microtubules (red) are attached to coverslip surface. Short non-biotinilated microtubules (cyan) are cross-linked to the red microtubules by Ase1 crosslinkers (not labeled).
www_video3_ncd_Ase1_sliding_edited Ase1 crosslinkers prevent sliding microtubules from separation. Long biotinilated microtubule (dim red) is bound to the coverslip surface, while a shorter non-biotinilated microtubule (bright red) is cross-linked to the biotinilated one by Ase1-GFP (green) and kinesin-14 (not labeled). Microtubule sliding is driven by kinesin-14 molecular motors. Ase1-GFP diffuse in microtubule overlaps, with the overlap ends forming a diffusion barrier. Once the microtubules start to separate, the converging overlap ends herd the Ase1-GFP molecules in the shortening overlap and the microtubule sliding stalls.

Head of Group

Zdeněk Lánský

Lansky_Zdenek_MPI_CBG

Profile

Most Significant Recent Publications

Lansky Z, Braun M, Lüdecke A, Schlierf M, ten Wolde PR, Janson ME, Diez S. Diffusible Crosslinkers Generate Directed Forces in Microtubule Networks. Cell 2015, 160: 1159–1168
See also Preview by David Odde, Cell 2015, 160: 1041-43

Braun M, Lansky Z, Fink G, Ruhnow F, Diez S, Janson ME. Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart. Nature Cell Biology 2011, 13(10): 1259-64

Verbrugge S, Lansky Z, Peterman EJG. Kinesin’s step dissected with single motor FRET. PNAS 2009, 106(42): 17741-6

Group Profile (Home Institution)

Publications

Braun M, Diez S, Lansky Z. Cell Biology: Kinesin-14 Backsteps to Organize Polymerizing Microtubules. Curr Biol 2016, 26(24):R1292-R1294.

Korten, T, Chaudhuri S, Tavkin E, Braun M, Diez S, Kinesin-1 Expressed in Insect Cells Improves Microtubule in Vitro Gliding Performance, Long-Term Stability and Guiding Efficiency in Nanostructures, IEEE Trans Nanobioscience 2016, 15:62–69.

Jirku M, Lansky Z, Bednarova L, Sulc M, Monincova L, Majer P, Vyklicky L, Vondrasek J, Teisinger J, Bousova K. The characterization of a novel S100A1 binding site in the N-terminus of TRPM1. Int J Biochem Cell Biol 2016; 78:186-93.

Braun M*, Lansky Z*, Hilitski F, Dogic Z, Diez S. Entropic forces drive contraction of cytoskeletal networks. Bioessays 2016, 38(5):474-81.

Lansky Z*, Braun M*, Lüdecke A, Schlierf M, ten Wolde PR, Janson ME, Diez S. Diffusible Crosslinkers Generate Directed Forces in Microtubule Networks. Cell 2015, 160: 1159–1168
See also preview by David Odde, Cell 2015, 160(6): 1041-43

Grycova L, Holendova B, Lansky Z, Bumba L, Jirku M, Bousova K, Teisinger J. Ca2+ binding protein S100A1 competes with calmodulin and PIP2 for binding site on the c-terminus of TRPV1 receptor. ACS Chem Neurosci 2014, 6(3):386-92.

Braun M*, Lansky Z*, Bajer S, Fink G, Kasprzak AA, Diez S. The human kinesin-14 HSET tracks the tips of growing microtubules in vitro. Cytoskeleton 2013, 70(9):515-21

Grycova L, Holendova B, Bumba L, Bily J, Jirku M, Lansky Z, Teisinger J. Integrative Binding Sites within Intracellular Termini of TRPV1 Receptor. PLoS ONE 2012, 7(10): e48437.

Braun M*, Lansky Z*, Fink G, Ruhnow F, Diez S, Janson ME. Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart. Nat Cell Biol 2011, 13(10):1259-64

Lansky Z, Peterman EJG. Studying kinesin’s enzymatic cycle using a single-motor confocal motility assay, employing Förster resonance energy transfer. Methods Mol Biol 2011, 778:19-32.

Verbrugge S*, Lansky Z*, Peterman EJG. Kinesin’s step dissected with single motor FRET. Proc Natl Acad Sci USA 2009, 106(42):17741-6

Kubala M, Grycova L, Lansky Z, Otyepka M, Amler E, Teisinger J. Changes in electrostatic potential induced by ATP binding to Na+/K+-ATPase Cytoplasmic Headpiece. Biophys J 2009, 16;97(6):1756-64.

Grycova L, Sklenovsky P, Lansky Z, Otyepka M, Amler E, Teisinger J, Kubala M. ATP and Magnesium Drive Conformational Changes of the Na+/K+-ATPase Cytoplasmic Headpiece. Biochim. Biophys. Acta 2009, 1788(5): 1081-1091.

Grycova L, Lansky Z, Vlachova V, Obsil T, Teisinger J. Ionic interactions are essential for TRPV1 C-terminus binding to calmodulin. Biochem Biophys Res Commun 2008, 375(4): 680-683

Grycova L. Lansky Z, Friedlova E, Vlachova V, Kubala M, Obsil T, Teisinger J. ATP binding site on the C-terminus of the vanilloid receptor. Arch Biochem Biophys 2007, 465(2): 389-398

Handl M, Filova E, Kubala M, Lansky Z, Kolacna L, Vorlicek J, Trc T, Pach M, Amler E. Fluorescent advanced glycation end products in the detection of factual stages of cartilage degeneration. Physiol Res. 2007; 56(2):235-42.

Krumscheid R, Ettrich R, Susankova K, Lansky Z, Hofbauerova K, Linnertz H, Teisinger J, Amler E, Schoner W. The phosphatase activity of the isolated H4-H5 loop of Na+/K+-ATPase resides outside of its ATP site. Eur J Biochem 2004, 271: 3923-3936.

Lansky Z, Kubala M, Ettrich R, Kuty M, Plasek J, Teisinger J, Schoner W, Amler E. The hydrogen bond between Arg423 and Glu472 and another key residues Asp443, Ser477 and Pro489 are responsible for forming and different positioning of TNP-ATP and ATP within the nucleotide binding site of Na+/K+-ATPase. Biochemistry 2004, 43: 8303-8311

Kubala M, Obsil T, Obsilova V, Lansky Z, Amler E. Protein modeling combined with spectroscopic techniques: an attractive quick alternative to obtain structural information. Physiol Res. 2004, 53 Suppl 1:S187-97.

* equal contribution

 

Projects

1) Entropy and contractile forces

Molecular motors are the canonical force generators in cytoskeleton. They generate forces by harnessing energy stored in chemical bonds, for example by hydrolyzing ATP. Other, cytoskeletal proteins, which cannot hydrolyze ATP, can generate forces of the same magnitude by harnessing the thermal energy of their environment. Examples of the latter are the members of the Ase1/PRC1/Map65 protein family of microtubule cross-linkers. These cross-linkers bind with their two binding sites diffusively between two microtubules. They have rather low unbinding rate from the microtubules, which effectively traps them once they bind into the microtubule overlap. The diffusible cross-linkers confined in the overlap then generate an entropic force, which acts to increase the length of the overlap (volume occupied by the cross-linkers). This mechanism is analogous to gas particles enclosed by a piston in a cylinder generating pressure, which moves the piston such that the volume occupied by the particles increases. By increasing the length of the microtubule overlap, these Ase1 cross-linkers generate contractile force in the microtubule network.

Further reading:
Lansky Z et al, Diffusible crosslinkers generate directed forces in microtubule networks, Cell 2015, 160(6): 1159-68

Braun M et al, Entropic forces drive contraction of cytoskeletal networks, Bioessays 2016, 38(5): 474-81

Odde DJ, Mitosis, diffusible crosslinkers, and the ideal gas law, Cell 2015, 160(6): 1041-3.

2) Regulation of kinesin motor proteins

Microtubule-crosslinking molecular motors, such as the members of the kinesin-14 family, remodel microtubule networks by sliding antiparallel microtubules along each other. To maintain the integrity of the network and to prevent the microtubules from sliding apart completely, the sliding has to be regulated. This regulation can be performed by i) additional proteins. The members of the Ase1/PRC1/Map65 protein family of microtubule cross-linkers can bind into the antiparallel overlaps between two microtubules propelled by kinesin motors. When the D. melanogaster kinesin-14, Ncd, motors start sliding two microtubules apart, the amount of Ase1 in the shortening overlaps stays approximately constant due to its high affinity for the overlap region (see above), whereas the amount of the Ncd motors decreases with the decreasing overlap length. This leads to less and less motors working against the same amount of Ase1 cross-linkers, which results in a decrease in the sliding velocity. Additionally, Ase1 generates entropic force acting against the motor-driven decrease in the overlap length (see above). ii) The regulation can be also performed by the motors themselves. Human kinesin-14, HSET, slides antiparallel microtubules and, due to the specific binding kinetics of its two microtubule binding sites, can diffuse in the microtubule overlap and is confined therein. When HSET start sliding two microtubules apart, its amount in the overlap stays approximately constant leading to an increase in its density. Increase in the HSET density then results in a decrease in sliding velocity through density-dependent decrease in the driving force, likely due to steric hindrance between the motors and the generation of an entropic force acting against the ATP-dependent motor-generated force driving the microtubules apart.
Both mechanisms of motor regulation act as an adaptive braking system, enabling the transport of fully overlapping microtubules but leading to the decrease in sliding velocity and the formation of stable overlaps once the microtubules start sliding apart.

Further reading:
Braun M et al, Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart, Nat Cell Biol, 2011, 13(10): 1259-64

Team

lansky_group

Zdeněk Lánský

  • Head of group

Puttrich Verena

Marcus Braun

Ondřej Kučera

Ilia Zhernov

Silvie Svidenská

Radan Matura

Roman Podhájecký

Hana Vratislavská

Eva Masárová

Contacts

Institute of Biotechnology of the ASCR, v.v.i., BIOCEV
Průmyslová 595
252 50 Vestec near Prague
Czech Republic
+420 325 873 772
Zdeněk Lánský zdenek.lansky@ibt.cas.cz +420 325 873 772
Puttrich Verena verena.puttrich@ibt.cas.cz +420 325 873 773
Marcus Braun marcus.braun@ibt.cas.cz +420 325 873 772
Ondřej Kučera ondrej.kucera@ibt.cas.cz +420 325 873 772
Ilia Zhernov ilia.zhernov@ibt.cas.cz +420 325 873 772
Silvie Svidenská silvie.svidenska@ibt.cas.cz +420 325 873 772
Radan Matura radan.matura@ibt.cas.cz +420 325 873 772
Roman Podhájecký roman.podhajecky@natur.cuni.cz +420 325 873 772
Hana Vratislavská +420 325 873 772
Eva Masárová +420 325 873 772