Microtubules are long filaments made up from protein building blocks and ubiquitously employed by eukaryotic cells for a wide range of often essential cellular processes. To perform these functions, microtubules are virtually always organized into higher order networks. Microtubule networks in cells of land plants are fundamental for guiding growth processes and for bringing about their unique mode of cell division. The latter is facilitated by the so‑called phragmoplast network, consisting of two opposing sets of microtubules that foster in their centre the formation and radial outgrowth of a disc-shaped membrane compartment (termed the cell plate) that ultimately divides the two daughter cells. The mechanisms driving the spatial organisation of such networks are of outstanding interest because plant cells do not rely on major microtubule organizers as in most other organisms. Instead, plant cells use a wide range of dispersed interactions among individual microtubules to shape functional microtubule networks. Chapter 1 introduces encounters between microtubules of opposite polarity and consequent bundling as potentially powerful handles to organize microtubules into networks. These encounters generate an area of antiparallel microtubule overlap and such overlaps are a striking feature of the phragmoplast microtubule network.
For long it is recognized that the short overlaps formed among the two opposing sets of phragmoplast microtubules and the membranous structures of the cell plate fall within the same plane. In chapter 2 we hypothesize that the limited length of these overlaps is required for the confined accumulation of cell plate membranes. To investigate this, we start out by co-visualizing overlaps and cell-plate membrane material in living cells of the moss Physcomitrella patens, an emerging model plant system with a convenient genetic toolset and tissues readily observable through microscopy. We reaffirm an early association between overlaps and membranes and further explored this association by experimentally altering overlap length. Incited by length control mechanisms of overlaps in animal cells, we identify two kinesin-4 motor proteins that jointly limit the length of phragmoplast microtubule overlaps in moss. Using cells lacking these kinesin-4s we then show that over-elongation of microtubule overlaps leads to a broadening of initial cell plate membrane depositions and a delayed progression of radial cell plate outgrowth. The cross walls ultimately formed by the wider membrane depositions were found to be thick and irregularly shaped. We thus demonstrate that kinesin-4-dependent overlap shortening in the phragmoplast defines the site of cell plate synthesis for the proper scaffolding of a new cell wall segment separating two daughter cells.
In chapter 3 we further investigate molecular mechanisms that could explain how linkage between a microtubule overlap and membrane assembly activity is realized. We focus on the exocyst tethering complex, one of the membrane tethering complexes involved in cell plate formation in flowering plants. We survey the localization of several moss exocyst subunits during cell division and find that one (Sec6) localizes to microtubule overlaps already before the onset of cell plate biogenesis. Experiments in which overlap length is altered and overlap formation is suppressed reveal that these structures play an important role in positioning Sec6 during cell division. The ability of moss Sec6 to interact with an evolutionary conserved factor in cell plate membrane fusion called KEULE is demonstrated, signifying a potential functional link between membrane tethering and fusion activities during cell plate formation. The precise role of Sec6 positioning by overlaps is as yet unclear, but in the light of the importance of overlaps for spatial control of cytokinesis will prove to be an intriguing direction for future research efforts.
In chapter 4 we gain further mechanistic insight in kinesin-4 mediated overlap length control and governance of division apparatus length as a whole. We focus on microtubule growth in overlaps regulated by kinesin-4, the poleward transport of microtubule polymers (termed flux), and the interplay between these processes. First, a method based on localized photo-activation is established for the quantitative assessment of microtubule flux. We demonstrate that initially flux in the metaphase spindle occurs synchronized and at high rates, to be replaced by a heterogeneous and on average much slower microtubule flux in the phragmoplast. Since polymerisation of microtubules could provide direct fuel for flux, we postulate that the rate of microtubule growth at sites of overlap could determine flux rates. To test this, we experimentally enhance polymerisation rates through knock-out of kinesin-4 proteins. This approach is validated by experiments demonstrating that they can supress microtubule outgrowth at overlaps in an in vivo setting. Upon kinesin-4 removal, flux rates are enhanced signifying coupling to rates of polymerization. We also find that lack of kinesin-4s leads elongation of the entire division apparatus and that this length change is proportional to the temporal activity patterns of the two kinesin-4s. Based on these findings we propose a mechanism for length regulation through a balance of microtubule growth in the overlap zone, retrograde microtubule translocation and putatively microtubule breakdown at the poles. Microtubule turnover in this system is high in the metaphase spindle (~1.5 μm/min), which, partly through kinesin-4 action, is succeeded by a more slowly turning over system in the form of the phragmoplast.
While in general the involvement of antiparallel microtubule overlaps in spatial organization of bipolar microtubule configurations is evident, how they could help shape other geometries is largely unknown. Chapter 5 starts out with the observation that within the unipolarized microtubule array of tip growing moss cells during interphase, there is occasional formation of overlaps at dispersed sites in the network. Tip growth is a mode of growth allowing rapid colonization of the environment and is achieved through highly polarized secretion, whereby the microtubule network reportedly steers the grows axis. We identify one kinesin-4 motor (Kin4-Ia) recruited to the observed overlaps within this network and use knock-out of Kin4-Ia to assess its role in tip growth. This reveals that absence of Kin4-Ia leads to a less adaptable axis of tip growth, prompting further investigation of Kin4-Ia behaviour at interphase overlaps. We find that this kinesin-4 is recruited with a slight delay to overlaps after their formation and inhibits plus end polymerization of overlap microtubules, thereby limiting overlap length. We then uncover that this activity helps to keep the network polarized towards the tip and prevent the overall organization from becoming hyperaligned with the cell axis. We propose that the latter observation might explain the decrease in growth axis adaptability. Overall, this thesis demonstrates that in plant microtubule networks of varying architecture, the formation of antiparallel overlaps provides a defined network feature for the recruitment of other microtubule-based process. Together, overlaps and activities coordinated from there, are potent organizers of functional plant microtubule arrays. The potential wider implications of these findings, their relationship to membrane-bound cytokinetic processes, and their evolutionary context are briefly discussed in Chapter 6.