For each living organism health is ensured by correct functioning of its cells. Cells therefore have elaborate methods for regulation of their proteins. In this thesis we examine a set of highly... Show moreFor each living organism health is ensured by correct functioning of its cells. Cells therefore have elaborate methods for regulation of their proteins. In this thesis we examine a set of highly conserved proteins: microtubules (MTs), which are either in a growing or a shrinking phase, together with the MTs' end-binding proteins mal3, tea2 and tip1. Using total internal reflection fluorescence microscopy we grow these proteins in custom-made, patterned flow chambers. We also examine the influence of force on MTs by letting them grow against obstacles, using the same microscopy technique or optical tweezers.Focusing on the switch from a growing to a shrinking state, a so-called catastrophe, we investigate the trigger of a catastrophe. For this we create distributions of the length of MT growth phases and fit them with several functions. All of the functions describe reactions involving several steps. We find that MTs need two steps until a catastrophe, a process which is sped up by the end-binding proteins as well as by force. Besides, we also present another model for MT catastrophe in which noisy growth together with a specific state of the microtubule tip are capable to explain a lot of the published MT data. Show less
Microtubuli (MT) zijn holle buisjes opgebouwd uit eiwit tubuline. Ze zijn meest bekend van hun rol in de celdeling, waarbij ze het genetisch materiaal verdelen over de twee dochtercellen. In een... Show moreMicrotubuli (MT) zijn holle buisjes opgebouwd uit eiwit tubuline. Ze zijn meest bekend van hun rol in de celdeling, waarbij ze het genetisch materiaal verdelen over de twee dochtercellen. In een niet delende cell vormen deze buisjes een netwerk door de cel heen en zijn betrokken bij verschillende processen, o.a. transport, cel polarisatie en celmigratie. Microtubuli groeien en krimpen aan hun uiteindes. Het meest dynamische uiteinde wordt ook het plus__eind genoemd. In dit onderzoek hebben we gekeken hoe deze plus__eindjes eruit zien als ze krimpen/ groeien. Zo hebben we mbv een electronen microscoop ingezoemd op de plus__eindjes van de microtubuli en deze in detail beschreven. We vonden negen verschillende vormen die plus__eindjes kunnen aannemen. Met behulp van een truuk hebben we uitgezocht welke vorm van de plus__eindjes behoort bij de krimpende of de groeiende en hoe de transitie van de groei naar de krimp verloopt. Onze resultaten hebben ook uitgewezen dat bepaalde plus__eindjes door de factoren in de cel vastgehouden kunnen worden. Dit betekent dat de structuur van de plus__eind belangrijk zou kunnen zijn voor de interacties met verschillende organellen. Kennis over deze interacties is van belang bij de begrijpen van de processen als celdeling, waarbij de plus__eindjes interactie met het genetisch materiaal aangaan. Show less
Microtubules are highly dynamic protein polymers that and are essential for intracellular organization and fundamental processes like transport and cell division. In cells, a wide family of... Show moreMicrotubules are highly dynamic protein polymers that and are essential for intracellular organization and fundamental processes like transport and cell division. In cells, a wide family of microtubule-associated proteins (MAPs) tightly regulates microtubule dynamics. The work presented in this thesis gives a high-resolution perspective on the microtubule assembly process and on the regulation mechanisms employed by representative MAPs. We studied dynamic microtubules outside cells, in a reconstituted minimal system. To follow microtubule growth with near molecular resolution, we developed a high-resolution technique that integrates optical tweezers, micro-fabricated rigid barriers and high-resolution video tracking of microbeads. Using this technique we found, for example, that microtubule assembly does not always occur by addition of single protein subunits, but multiple subunits could be incorporated at once at the growing end. XMAP215, a protein known to dramatically enhance microtubule growth, altered these molecular details. Another intriguing protein studied here is Mal3, a protein that is able to track growing microtubule ends. We found that Mal3 interacts differentially at the growing tip and on the rest of the microtubule, influencing all the parameters describing microtubule dynamics. In conclusion, our results give new insights into the microtubule assembly process in the absence and in the presence of regulators. Show less
