Building Minimal Spindles
Reconstituting spindle positioning in synthetic cells
Y.K. Jawale (TU Delft - BN/Marileen Dogterom Lab)
A.M. Dogterom – Promotor (TU Delft - BN/Marileen Dogterom Lab)
L. Laan – Promotor (TU Delft - BN/Liedewij Laan Lab)
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Abstract
The cell is the fundamental unit of life, composed of smaller, non-living components. This raises a key question: what truly gives rise to life? This thesis explores cytoskeletal organization and dynamics, focusing on microtubules and spindle positioning, with the broader aim of reconstituting these processes in synthetic cells.
Encapsulation of biological components is central to synthetic cell research. We evaluated different methods to create cell-like compartments, finding that while droplets are easy to work with, cDICE offers greater flexibility for functional encapsulation. Using high-speed imaging, we studied GUV formation in cDICE and discovered a size-selective crossing of droplets at the interface. We also found that proteins in the inner solution affect GUV formation by increasing viscosity and altering lipid adsorption.
Tubulin, an essential protein in cells, remains difficult to work with in vitro. Using different encapsulation methods, we observed that tubulin influences the stability of lipid bilayers, and using a membrane interaction assay, we found that it can even disrupt the membranes.
To increase biological relevance, we combined major cellular components like microtubule asters with an actin cortex or a nucleus mimic, and explored external tools for spatiotemporal control. We successfully assembled a mitotic spindle-like organization in droplets. We incorporated an optogenetic switch to control dynein in order to achieve asymmetric spindle positioning, inspired by the first cell division of the C. elegans embryo. However, light-induced transport remains limited.
Finally, we adapted the bacterial ParMRC DNA segregation system for synthetic cell, with light-regulated control via iLID. Although individual components react to light activation, further optimization is needed to make the full system responsive.
These reconstitutions provide insight into fundamental mechanisms of spindle positioning and are basic steps toward building a minimal synthetic spindle.
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