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

AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into howthese factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. Wethus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.Plant science

Function, localization and evolution of SOSEKI polar proteins

The evolution of multi-cellular plants went hand in hand with the establishment of a complex polarity system to guide development and survival. Within the cell, polarity cues need to be established, read and translated into sub-cellular processes. Yet, the exact mechanisms that translate polarity into sub-cellular processes remain elusive. In Chapter 1, we discuss polarity and several proteins that use polar information to guide their localization. The Arabidopsis embryo is introduced as an excellent model for studying cell polarity. In Chapter 2, we take a closer look at development of the Arabidopsis embryo. Hereby we focus specifically on how oriented divisions are generated by developmental regulators and the division machinery. Recent advancement in 3D imaging of the embryo revealed that cell division abides to a ‘smallest plane’ rule, and that auxin can prevent adherence to this rule. Studying how auxin effectors are linked to cell division regulators and cell polarity may provide a greater understanding of oriented cell division in the embryo. Using the Arabidopsis embryo as model for auxin-regulated development, we identify a novel family of polarly localized proteins in Chapter 3. Unlike previously published polar proteins, this new family shows a robust localization to specific cell edges, which coined the name SOSEKI (SOK, Japanese for cornerstone). SOK localization is guided by integration of plant-wide apico-basal and radial polarity. Pharmacological inhibition of pathways commonly used by polarly localized proteins showed that SOK is localized through a novel mechanism. Mis-expression of SOK1 caused oblique cell divisions and polar localization was required for this activity. We identified a highly conserved N-terminal domain that structurally resembles the DIX domain found in Wnt polarity signalling proteins in animals (Ehebauer & Arias, 2009; Schwarz-Romond et al., 2007). In animals, this domain shows autocatalytic polymerization. SOK1 DIX-LIKE can dimerize and is required for polar edge clustering and biological activity, which shows that the fundamental function of DIX is conserved. Taken together, this chapter revealed a compass of polar axes that guides SOK polar edge localization. In addition, we showed that both plants and animals use the DIX domain in the context of polarity. SOK showed striking localization and behavior, but nothing was known about the function of this protein family. In Chapter 4, we studied SOK function by generating sok mutants. We found that small mutations near the N-terminal end of SOK1 sometimes caused fertility defects, but that larger deletions had no effect. The sok1 deletion mutant showed upregulation of the SOK4 gene, which suggests that there may be a compensation mechanism or feedback loop. The potential redundancy between SOK1 and SOK4 led to further investigation of SOK expression and localization throughout the plant. Based on our findings, SOK2 and 3 may be redundant in the leaf, while SOK2, 3 and 5 overlap in the gynoecium. As SOK was a completely novel protein family with unknown origin, we aimed to learn more about its evolutionary history. Therefore we investigated the protein sequence, properties and polar localization throughout plant evolution in Chapter 5. We showed that SOK first arose in early land plants, and that they contain several conserved domains that separate SOKs in an ancestral and a more recently evolved type. To assess the conservation of polarity, we studied four SOKs in the moss Physcomitrella patens. One of these tested PpSOKs showed polar edge accumulation in the gametophore, which suggests that edge polarity of SOK proteins is conserved throughout evolution. Next we performed phylogenetic and functional analysis on the DIX domain, which is the most highly conserved domain of SOK. Our results revealed that DIX is present in land plants, animals and the SAR group, and that it is capable of polymerization in all these clades. The molecular context of a protein can reveal how it functions within the cell and how it obtains its localization. To address these questions in Chapter 6, we combined biochemistry and cell biology and identified shared and unique interactors of SOK1, SOK2 and SOK3. At least one of these interactors was recruited to the polar SOK1 site in a DIX-LIKE-dependent manner. We extended the network of interaction partners and found that SOK1 interacts with a network of laterally-polar proteins. The secondary interactors revealed links with amongst others the cytoskeleton. Based on these findings, we propose that DIX-like-mediated polymerization creates a polar scaffold that recruits interactors for local tasks. Such tasks may be modification of the cytoskeleton during cell growth or mechanical stress. To conclude this thesis, the context and implications of our results were discussed in Chapter 7. In this discussion, we also provide an outlook for the future and suggestions for application of our results in research and biotechnology.</p

Using a Caenorhabditis elegans Parkinson’s Disease Model to Assess Disease Progression and Therapy Efficiency

Despite Parkinson’s Disease (PD) being the second most common neurodegenerative disease, treatment options are limited. Consequently, there is an urgent need to identify and screen new therapeutic compounds that slow or reverse the pathology of PD. Unfortunately, few new therapeutics are being produced, partly due to the low throughput and/or poor predictability of the currently used model organisms and in vivo screening methods. Our objective was to develop a simple and affordable platform for drug screening utilizing the nematode Caenorhabditis elegans. The effect of Levodopa, the “Gold standard” of PD treatment, was explored in nematodes expressing the disease-causing α-synuclein protein. We focused on two key hallmarks of PD: plaque formation and mobility. Exposure to Levodopa ameliorated the mobility defect in C. elegans, similar to people living with PD who take the drug. Further, long-term Levodopa exposure was not detrimental to lifespan. This C. elegans-based method was used to screen a selection of small-molecule drugs for an impact on α-synuclein aggregation and mobility, identifying several promising compounds worthy of further investigation, most notably Ambroxol. The simple methodology means it can be adopted in many labs to pre-screen candidate compounds for a positive impact on disease progression

Using a Caenorhabditis elegans Parkinson&rsquo;s Disease Model to Assess Disease Progression and Therapy Efficiency

Despite Parkinson&rsquo;s Disease (PD) being the second most common neurodegenerative disease, treatment options are limited. Consequently, there is an urgent need to identify and screen new therapeutic compounds that slow or reverse the pathology of PD. Unfortunately, few new therapeutics are being produced, partly due to the low throughput and/or poor predictability of the currently used model organisms and in vivo screening methods. Our objective was to develop a simple and affordable platform for drug screening utilizing the nematode Caenorhabditis elegans. The effect of Levodopa, the &ldquo;Gold standard&rdquo; of PD treatment, was explored in nematodes expressing the disease-causing &alpha;-synuclein protein. We focused on two key hallmarks of PD: plaque formation and mobility. Exposure to Levodopa ameliorated the mobility defect in C. elegans, similar to people living with PD who take the drug. Further, long-term Levodopa exposure was not detrimental to lifespan. This C. elegans-based method was used to screen a selection of small-molecule drugs for an impact on &alpha;-synuclein aggregation and mobility, identifying several promising compounds worthy of further investigation, most notably Ambroxol. The simple methodology means it can be adopted in many labs to pre-screen candidate compounds for a positive impact on disease progression

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division 1–3 . In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI proteins that integrate apical–basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. A Domain of Unknown Function in SOSEKI proteins resembles the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation, together with a second domain that defines the polar membrane domain. Our work shows that SOSEKI proteins locally interpret global polarity cues and can influence cell division orientation. Furthermore, this work reveals that, despite fundamental differences, cell polarity mechanisms in plants and animals converge on a similar protein domain. </p

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division 1–3 . In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI proteins that integrate apical–basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. A Domain of Unknown Function in SOSEKI proteins resembles the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation, together with a second domain that defines the polar membrane domain. Our work shows that SOSEKI proteins locally interpret global polarity cues and can influence cell division orientation. Furthermore, this work reveals that, despite fundamental differences, cell polarity mechanisms in plants and animals converge on a similar protein domain. </p

AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development