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Research

The Grebe research group

We investigate how epidermal cell and tissue polarity is established in plants mostly using the root epidermis of Arabidopsis thaliana as a model system. We combine genetic, cell biological, physiological, biochemical and molecular biology methods to understand how at the molecular level one end of the cell becomes different from another and how this is coordinated within the tissue context.

Planar polarity in the Arabidopsis root epidermis

In this project, we originally addressed the role of the plant hormone auxin in coordinating cell polarity within the plane of the root epidermal layer (planar polarity) upstream of the coordinated polar positioning of Rho-of-plant (ROP) GTPases at the site of root hair initiation (Fischer et al. 2006; Ikeda et al. 2009). In genetic screens and protein-protein interaction experiments, we then identified modulators of cytoskeletal organization during planar polarity formation. These included, among others, the SABRE and the AIP1-2 protein (Pietra et al. 2013; Kiefer et al. 2015), whose function we are currently further investigating in ongoing projects. In this context, organization and dynamics of the actin and tubulin cytoskeletons during cell and planar polarity formation are analyzed by high-resolution quantitative live cell imaging. Their regulation is dissected by combined genetic, molecular, biochemical and modeling approaches.

Outer lateral epidermal polarity in the Arabidopsis root

This project addresses how membrane proteins polarly localized at the outer epidermal membrane domain facing the soil, such as the PENETRATION3 (PEN3) ABCG transporter protein, are trafficked to and from this domain and how their polarity is established (Mao et al. 2016). We recently identified a novel BTB/POZ-domain protein specifically contributing to the trafficking of PEN3 from the endoplasmic reticulum (Mao et al. 2017). We are currently further addressing its cellular and molecular functions as well as interactions.

Polar nuclear migration into root epidermal hair cells

This project addresses how different polar nuclear migration events in root epidermal hair cells are regulated by auxin, ROP signaling and the cytoskeleton, employing a combination of cell-type specific modulation of the cytoskeleton and its regulators with live cell imaging of nuclear migration (Nakamura et al. 2018). In this project, we have also identified regulators of ROPs as being responsible for the directed movement of the nucleus into the root hair (Nakamura et al. 2018) and investigated the role of ROPs and their regulators in root hair elongation (Gendre et al. 2019). We are currently further investigating their role in planar polarity formation.

Sterol and proteo-lipid domain function during cytokinesis and epidermal cell polarity

This project studies the role of sterols and proteo-lipid domains during cytokinesis and polar root hair initiation (Men et al. 2008; Boutté et al. 2010, Stanislas et al. 2015). High-resolution fluorescence imaging of sterols as well as ratiometric imaging of lipid-order sensitive probes are combined with genetic and pharmacological tools for sterol and lipid interference to address their functions during cell plate and cell polarity formation.

Selected publications:

Gendre D, Baral A, Dang X, Esnay N, Boutté Y, Stanislas T, Vain T, Claverol S, Gustavsson A, Lin D, Grebe M, Bhalerao RP (2019). Rho-of-plant activated root hair formation requires Arabidopsis YIP4a/b gene function. Development 146: pii: dev168559. doi: 10.1242/dev.168559.

Nakamura M, Claes AR, Grebe T, Hermkes R, Viotti C, Ikeda Y, Grebe M (2018). Auxin and ROP GTPase signaling of polar nuclear migration in root epidermal hair cells. Plant Physiology 17: 378-391.

Mao H, Aryal B, Langenecker T, Hagmann J, Geisler M, Grebe M (2017). Arabidopsis BTB/POZ protein-dependent PENETRATION3 trafficking and disease susceptibility. Nature Plants 3: 854-858.

Mao H, Nakamura M, Viotti C, Grebe M (2016). A framework for lateral membrane trafficking and polar tethering of the PEN3 ATP-binding cassette transporter. Plant Physiology172: 2245-2260.

Stanislas T, Hüser A, Barbosa IC, Kiefer CS, Brackmann K, Pietra S, Gustavsson A, Zourelidou M,  Schwechheimer C, Grebe M (2015). Arabidopsis D6PK is a lipid domain-dependent mediator of root epidermal planar polarity. Nature Plants 1: 15162.

Kiefer CS, Claes AR, Nzayisenga JC, Pietra S, Stanislas T, Hüser A, Ikeda Y, Grebe M (2015). Arabidopsis AIP1-2 restricted by WER-mediated patterning modulates planar polarity. Development142: 151-161.

Pietra S, Gustavsson A, Kiefer C, Kalmbach L, Hörstedt P, Ikeda Y, Stepanova AN, Alonso JM, Grebe M. (2013). Arabidopsis SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity. Nature Communications 4: 2779.

Boutté, Y, Frescatada-Rosa, M, Men, S, Chow, CM, Ebine, K, Gustavsson, A, Johansson, L, Ueda, T, Moore, I, Jürgens, G, Grebe, M (2010). Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis. EMBO Journal 29: 546-558.

Ikeda Y, Men S, Fischer U, Stepanova AN, Alonso JM, Ljung K, Grebe M (2009). Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis. Nature Cell Biology 11: 731-738.

Men S, Boutté Y, Ikeda Y, Li X, Palme K, Stierhof YD, Hartmann MA, Moritz T, Grebe M (2008). Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nature Cell Biology10: 237-244.

Fischer U, Ikeda Y, Ljung K, Serralbo O, Singh M, Heidstra R, Palme K, Scheres B, Grebe M (2006). Vectorial information for Arabidopsis planar polarity is mediated by combined AUX1, EIN2, and GNOM activity. Current Biology 16: 2143-2149.

 

The Sauer research subgroup

We study the intracellular transport of proteins from their place of synthesis to the location where they function. Proteins are often transported in vesicles, small droplets surrounded by a biomembrane, which travel between subcellular compartments. We are particularly interested in factors that generate these vesicles and use cell biological, physiological and genetic methods to study them. Our research aims to get a detailed understanding of the composition and dynamics of the molecular machinery involved and what biological relevance it has for the plant as a whole.

Much of our research is centered around a group of proteins that contain specific functional domains, termed ENTH/ANTH/VHS (Zouhar and Sauer, 2014). For some members of this group, a role in intracellular transport and vesicle formation has been shown (Sauer et al. 2013, Heinze et al. 2020). However, the family is large and the biological role of many members is currently unclear.

Epsin-like proteins

In this project, we systematically analyze the four core ENTH proteins that most closely resemble Epsin proteins involved in generating clathrin coated vesicles (CCVs). We are particularly interested if these proteins associate with different molecular transport pathways or cargos and can be used to better distinguish previously unresolvable traffic routes.

Non-canonical ENTH proteins

Some proteins in plants contain a ENTH domain, but lack a large part of the interaction domains found in “classical” ENTH-type proteins. The function of these enigmatic ENTH proteins is currently unknown. In this project, we are trying to decipher the biological relevance and molecular mode of action of these proteins.

Environmental factors in protein transport

Plants need to cope with drastic changes of their environmental conditions. Parameters such as temperature, humidity, light levels, or nutrient availability vary strongly over time. We are interested whether environmental conditions affect intracellular transport and in which way. Ultimately, we want to find out if flexible responses of intracellular transport mechanisms are required to maintain plant vigor.

Selected publications:

Heinze L, Freimuth N, Rößling A-K, Hahnke, R, Riebschläger S, Fröhlich A, Sampathkumar A, McFarlane H E, Sauer M. (2020). EPSIN1 and MTV1 define functionally overlapping but molecularly distinct trans -Golgi network subdomains in Arabidopsis. PNAS 117: 25880–25889.

Delgadillo MO, Ruano G, Zouhar J, Sauer M, Shen J, Lazarova A, Sanmartín M, Lai LTF, Deng C, Wang P, Hussey PJ, Sánchez-Serrano JJ, Jiang L, Rojo E. (2020) MTV proteins unveil ER- and microtubule-associated compartments in the plant vacuolar trafficking pathway. PNAS 117: 9884-9895.

Zouhar J, Sauer M. (2014) Helping hands for budding prospects: ENTH/ANTH/VHS accessory proteins in endocytosis, vacuolar transport, and secretion. Plant Cell 26: 4232-4244.

Sauer M, Delgadillo MO, Zouhar J, Reynolds GD, Pennington JG, Jiang L, Liljegren SJ, Stierhof YD, De Jaeger G, Otegui MS, Bednarek SY, Rojo E. (2013) MTV1 and MTV4 encode plant-specific ENTH and ARF GAP proteins that mediate clathrin-dependent trafficking of vacuolar cargo from the trans-Golgi network. Plant Cell 25: 2217-2235.