Microtubule number and length determine cellular shape and function in Plasmodium.

Benjamin Spreng,Marek Cyrklaff,Pintu Patra,Friedrich Frischknecht,Claudia Di Biagio,Hannah Fleckenstein,Ulrich S Schwarz,Madlen Benz,Patrick Kübler

doi:10.15252/embj.2018100984

Abstract

Microtubules are cytoskeletal filaments essential for many cellular processes, including establishment and maintenance of polarity, intracellular transport, division and migration. In most metazoan cells, the number and length of microtubules are highly variable, while they can be precisely defined in some protozoan organisms. However, in either case the significance of these two key parameters for cells is not known. Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two α‐tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that α‐tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit.

Highlights

Microtubules are cytoskeletal filaments formed as hollow cylinders from dimers of a-tubulin and b-tubulin (Olmsted & Borisy, 1973; Fojo, 2008)
The a2tubulin gene appears essential and cannot be deleted (Kooij et al, 2005), while we could readily delete the a1-tubulin gene in a wildtype (WT) background and in a parasite expressing GFP and mCherry as cytoplasmic markers at different life cycle stages (Fig EV1C and D). a1-tubulin gene deletion was attempted before without success (Kooij et al, 2005); the improved transfection methods since might have allowed us to generate a transgenic line without problems
No sporozoites were found in the midgut or salivary glands of the mosquito (Tables 1 and 2); a main defect seems to occur at the oocyst stage

Summary

Introduction

Microtubules are cytoskeletal filaments formed as hollow cylinders from dimers of a-tubulin and b-tubulin (Olmsted & Borisy, 1973; Fojo, 2008). While the number of microtubules in axonemes is fixed and their length is stable (Linck et al, 2014), these two key parameters are rarely investigated in cytoplasmic or spindle microtubules. Work in neurons showed that the number of cytoplasmic microtubule is different in neurite-forming axons or dendrites (Yu & Baas, 1994), with the axon containing about 10-fold more microtubules. Individual microtubule length varied over three orders of magnitude Owing to those large variations, the precise number and length of cytoplasmic microtubules are rarely taken into account or even investigated in most mammalian or model cells under study. The high rate of growth and shrinkage of microtubules (Mitchison & Kirschner, 1984; Goodson & Jonasson, 2018) make the determination of these parameters difficult if not meaningless as they constantly change. Number and length of spindle microtubules, can be fixed and are likely important for spindle function, as shown in fission yeast (Ward et al, 2014)

Methods

Results

Conclusion