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Biophysics of reconstituted cytoskeletal systems

Department                             Bionanoscience

Principal investigator          Marileen Dogterom

E-mail address                       m.dogterom@tudelft.nl

 

The interaction between the TubR-LOV2 protein, tubC centromeric DNA, and TubZ filaments

Supervisor: Beatriz Orozco, b.e.orozcomonroy@tudelft.nl

With the aim of building a DNA segregation mechanism for a synthetic cell using bacterial cytoskeleton filaments, this project consists of testing the interaction of the engineered light-switchable protein TubR-LOV2 (fusion of the adaptor protein TubR with the light-responsive LOV2 domain) with tubC centromeric sequences and TubZ bacterial cytoskeleton filaments. TIRF microscopy and image analysis will be employed to assess the interaction among these components and the dynamicity of the filaments. Additional experiments, such as mass photometry or gel electrophoresis, may be used in conjunction with microscopy to optimize experimental parameters like the ratio at which the components interact more efficiently.

Techniques

  • TIRF microscopy
  • Mass photometry

Further reading

Aylett, C.H.S. & Löwe, J. (2012). PNAS, 109(41), 16522-16527. DOI: 10.1073/pnas.1210899109.

Olivi, L., et al. (2021). Nature Communications, 12(1), 4531. DOI: 10.1038/s41467-021-24772-8.

 

Control aster positioning in droplets

Supervisor: Yash Jawale, y.k.jawale@tudelft.nl

Correct positioning of the (mitotic) spindle is an essential process in cell division, as its position plays an important role in determining the division plane and hence the size of the daughter cells. The spindle consists of two microtubule asters (growing from centrosomes) exerting forces.

You will attempt to learn how the microtubule aster positions inside a 3D cell-like confinement, and how the force balance changes in response to constraints of the environment and additional components.

Objectives:

  1. Encapsulate minimal asters inside a droplet;
  2. Quantify aster position as a function of protein concentration and droplet size;
  3. Study the effect of organelle-like structures, by adding crowding, LUVs or nucleus, and motor proteins;
  4. Simulate aster positioning.

Techniques

  • Microfluidics to encapsulate microtubule asters inside droplets
  • Advanced microscopy techniques and image analysis tools
  • Cytoskeleton simulation tools such as CytoSim to make predictions

Further reading

Roth, S., et al. (2019). bioRxiv. DOI: 10.1101/770602.

 

Spatio-temporal control of spindle positioning  (simulations)

Supervisor: Yash Jawale, y.k.jawale@tudelft.nl

Correct positioning of the (mitotic) spindle is an essential process in cell division. Although in vitro reconstitution methods provide much insight into spindle positioning, they are not sufficient to study the complete spectrum of force balances. We therefore turn to simulations to better understand spindle positioning and predict what affects the force balance.

Objectives:

  1. Simulate spindle assembly and monitor involved forces;
  2. Study the effects of cell shape and size;
  3. Study the effects of force generators (such as motor proteins) at the cortex and in the cytoplasm;
  4. Mimic events in the cell division process using spatial and temporal cues in the simulations;
  5. Find conditions for asymmetric spindle positioning.

Techniques

  • Simulations to answer research questions.
  • CytoSim (a cytoskeleton simulation tool).

Further reading

Roth, S., et al. (2019). bioRxiv. DOI: 10.1101/770602.

 

Controlling lipid liquid-liquid phase separation on membranes

Supervisor: Yash Jawale, y.k.jawale@tudelft.nl

Lipid domains play an important role in organizing proteins on the cell membrane. The cellular membrane consists of different lipids, and via interactions between the lipids, they can form clusters (domains) with each other and thus create lipid phase separation. As some proteins bind to specific lipids, the formation of lipid domains on membranes can be used to recruit and organize these proteins.

Objectives:

  1. Find and tune the different lipid types and their ratios required to create lipid phase separation on 2D membranes (and on 3D membranes);
  2. Optimize the size and number of lipid domains;
  3. Add spatial and temporal control on phase separation using light.

 

Reconstituting microtubule dynamics inside micro and nanochannels

Supervisor: Nemo Andrea, n.andrea@tudelft.nl 

We have developed microfluidic devices with micro and nanochannels that we want to use for liquid-phase EM. Not much is known about microtubule growth behaviour in extreme confinement (high surface to volume ratio) like in nanochannels. The project would involve investigating the growth dynamics of microtubules in micro and nanochannels and comparing them to macroscopic flowchannels that is already well established. Increasing the complexity of the reconstituion by studying effects of adding microtubule-binding proteins or other filaments (e.g. actin) would also be an option. The project would involve the reconstitution of the system (finding the right conditions and concentration), imaging the system on a fluorescence microscope, working with microfabricated chips and data analysis in Python or Julia.

Techniques

  • Fluorescence microscopy
  • In vitro reconstitution
  • Data analysis
  • Optional: data management with Git and Git-Annex.