Shaping plant microtubule networks via overlap formation

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.</p

Shortening of microtubule overlap regions defines membrane delivery sites during plant cytokinesis

© The Author(s), 2016. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Current Biology 27 (2017): 514-520, doi:10.1016/j.cub.2016.12.043.Different 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. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly. 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, we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.The work has been financially supported by HFSP grant RGP0026/2011 to MEJ and GG.2018-01-2

Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response

Influence of atmosphere, interparticle distance and support on the stability of silver on α-alumina for ethylene epoxidation

The stability of supported metal particles is an important parameter in heterogenous catalysis. For silver catalysts supported on α-alumina, industrially used in ethylene epoxidation, the loss of silver surface area as result of particle growth is one of the most important deactivation mechanisms. In this work, the growth of silver particles was investigated by exposing catalysts to thermal treatments. The presence of oxygen during heating strongly enhanced particle growth, and the interparticle distance was a crucial parameter. However, restricting movement of complete silver particles using cage-like α-alumina did not limit particle growth. These findings indicate that Ostwald ripening was the dominant mechanism behind particle growth, with the diffusion of oxidized silver species being a rate limiting factor. Finally, higher surface area α-alumina provided better silver stability during ethylene epoxidation, with only limited decrease in selectivity. This makes silver supported on high surface area α-alumina promising candidates for ethylene epoxidation catalysis

DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes

The identities of cell polarity determinants are not conserved between animals and plants; however, characterization of a DIX-domain containing protein in land plants reveals that the physical principles of polar complex assembly are preserved across eukaryotes.</p

Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Microtubule networks for plant cell division

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants

Physcomitrium patens : A single model to study oriented cell divisions in 1d to 3d patterning

Development in multicellular organisms relies on cell proliferation and specialization. In plants, both these processes critically depend on the spatial organization of cells within a tis-sue. Owing to an absence of significant cellular migration, the relative position of plant cells is virtually made permanent at the moment of division. Therefore, in numerous plant developmental contexts, the (divergent) developmental trajectories of daughter cells are dependent on division plane positioning in the parental cell. Prior to and throughout division, specific cellular processes inform, establish and execute division plane control. For studying these facets of division plane control, the moss Physcomitrium (Physcomitrella) patens has emerged as a suitable model system. Developmental progression in this organism starts out simple and transitions towards a body plan with a three-dimensional structure. The transition is accompanied by a series of divisions where cell fate transitions and division plane positioning go hand in hand. These divisions are experimentally highly tractable and accessible. In this review, we will highlight recently uncovered mechanisms, including polarity protein complexes and cytoskeletal structures, and transcriptional regulators, that are required for 1D to 3D body plan formation</p

Pore structure stabilization during the preparation of single phase ordered macroporous α-alumina

Ordered porous materials are highly relevant for applications in fields such as catalysis, adsorption and chromatography. Strategies such as soft and hard templating are now routinely applied to prepare for instance ordered mesoporous silica. In other cases, most notably when several polymorphic phase transformations are involved, achieving high quality ordered oxides is challenging. We present a strategy for the preparation of ordered macroporous α-alumina with a high degree of pore order. The preparation is based on impregnation of ordered polymeric spheres with an alumina precursor. Most notably it involves a first heating step in inert atmosphere, leading to conversion of the polymeric template into carbon effectively delaying the phase transitions and stabilizing the pore structure up to high temperatures. A subsequent heat treatment in oxidative atmosphere then removes the carbon and ordered marcoporous α-alumina is obtained with circa 220 nm cages interconnected by windows of circa 105 nm, and a specific surface area of circa 25 m2 g−1. Our method led to a strong preservation of the long-range order of the pore structure, as not only evidenced by electron microscopy, but also quantified by spectroscopy. High surface area α-alumina is of particular interest as a catalyst support, but the preparation method might also be extended to other ordered macroporous oxides that are difficult to prepare due to phase transitions such as TiO2