Shortening of Microtubule Overlap Regions Defines Membrane Delivery Sites during Plant Cytokinesis

Abstract

SummaryDifferent from animal cells that divide by constriction of the cortex inwards, cells of land plants divide by initiating a new cell wall segment from their centre. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands towards the parental cell wall [1][2][3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4][5][6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7][8][9]. How vesicle immobilisation and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell plate formation is lacking. Here we show that membranous material for cell plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4 mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins [10,11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis. Results and DiscussionEarly ultrastructural work in the flowering plant Haemanthus katherinae has revealed that microtubules from the two halves of the phragmoplast interdigitate at the midplane where cell plate vesicles aggregate [12]. The same configuration exists in the phragmoplast of the moss Physcomitrella patens [13], indicating that it might be universally relevant for the spatial orchestration of cytokinesis in land plants. We therefore investigated whether regions of antiparallel microtubule overlap could have a direct role in determining the location of cell plate assembly in the genetically amenable moss P. patens ( Figure 1A). For this we imaged the conserved microtubule bundling protein MAP65c labelled with citrine (a GFP variant) as a marker for microtubule overlaps [14][15][16] in combination with the lipophilic dye FM4-64 that stained the membrane of the forming cell plate in life caulonema apical cells ( Figure 1A Whether kinesin-4 has a direct role in division plane reorientation is currently unclear because the underlying process is mechanistically not understood.The mechanism by which kinesin-4 induces overlap shortening was investigated using a purified recombinant N-terminal fragment of moss Kin4-Ic. We observed that it supressed microtubule growth velocities in a dose dependent manner with a minimal effect on catastrophe rate ( Figure S2E-H Having found a way to increase overlap length, we next examined the consequenc...

Dual localized kinesin‐12 POK 2 plays multiple roles during cell division and interacts with MAP 65‐3

“…Comparing the signal distribution with GFP-POK2, we noticed that the signal of GFP-POK2 (1-589) at the phragmoplast midzone appeared wider (Figs 4G and EV1E and G, Appendix Fig S2A). Therefore, we quantified the average full width half maximum (FWHM) [40] of both signals at the midzone and confirmed that the distribution of GFP-POK2 (1-589) is considerably broader than GFP-POK2, while the phragmoplast size remains equal ( Fig 4H and I).…”

Control mechanisms of microtubule overlap regions

“…The interaction between CLASP and PRC1 suggests that a similar mechanism is present in animal cells (Liu et al, 2009). In higher eukaryotes kinesin-4 regulates the dynamics of microtubule overlaps additionally (Kurasawa et al, 2004;Bieling et al, 2007;Hu et al, 2011;de Keijzer et al, 2017). When overlap length decreases, the decrease in kinesin-4 will induce extra microtubule growth.…”

“…In the later stages of the animal spindle and in the plant phragmoplast, a regular array of short antiparallel overlaps is necessary for successful cytokinesis (Mollinari et al, 2002;Vernì et al, 2004;Zhu and Jiang, 2004;Hu et al, 2011;Kong et al, 2015;de Keijzer et al, 2017). The site of cytokinesis is directly linked to the site of overlaps.…”

“…In higher eukaryotes, PRC1 bundling proteins interact with kinesin-4 motors that slow microtubule growth. Kinesin-4 inhibits microtubule growth and its localization is regulated by PRC1, so it is mainly active on antiparallel overlaps (Kurasawa et al, 2004;Hu et al, 2011;de Keijzer et al, 2017). Also here, regulation is crucial and feedback between sliding and growth is required.…”

“…When overlap length is not regulated correctly, this causes problems in cell wall formation. In plants, the formation of longer overlaps results in irregular, thick cell walls, because vesicles with cell wall material are directed to the microtubule overlaps specifically (de Keijzer et al, 2017). In animal cytokinesis centralspindlin is involved in anchoring the microtubule overlaps to the plasma membrane (Lekomtsev et al, 2012).…”