Dr. Jan Maisch
Cell biology – pattern formation
Contrary to animals, plants lack the separation of immortal germ cells from mortal soma cells. In other words, each plant cell is basically a stem cell which can regenerate the entire organism. This ability is linked to an internal “direction” that can be traced down to a direction of each individual cell. Each plant cell is polarised – analogous to a small magnet. This cell polarity could drive a flow of a signal that drives patterning which is brought about by communication between cells that exchange morphogenetic signals in a certain direction. Since plant morphogenesis is open and characterised by indefinite growth this direction is continuously perpetuated by the cytoskeleton.
During development of an organism, the cytoskeleton is aligned in response to chemical signals by the plant hormone auxin. Auxin is transported directionally, an inter-cellular process that is related to actin-dependent vesicle traffic. It ensures the polar localisation of auxin-efflux carriers. This polar auxin transport defines what is "top" and "bottom" in a plant and how a pattern is established.
Auxin-signalling triggers the re-organisation of actin bundles into finer filaments. In turn, this enables more efficient transport of auxin-signalling components towards the cell pole. This will generate an oscillatory feedback loop (actin-auxin oscillator) where the actin configuration depends on auxin and auxin transport depends on the actin configuration.
How to analyze patterning?
Since it is very difficult to study pattern formation in whole plants or plant organs, we have to reduce complexity of the system. We work with tobacco cell cultures (Nicotiana tabacum L. cv. Bright Yellow 2, “BY-2”). These lines proceed from unicellular stages at the time of subcultivation through a series of axial cell divisions towards polar multi-cellular files. Another feature is that cell division follows a pattern which depends on polar auxin transport. The divisions within a file do not occur randomly, but are coordinated. The files represent a very simple multi-cellular organism where we can study how individual cells are coordinated into an entity - a fundamental question of developmental biology.
How does filamentous actin reorganise as reaction on the phytohormone auxin?
The signalling chain from auxin to actin during pattern formation remains unclear up to now. The motivation of our work is to shed light on this process by identification of potential auxin-signal mediators.
We used a genetic approach, where we expressed the actin-binding domain of mouse talin in fusion with YFP which led to constitutively bundled actin filaments. The synchrony of cell division was impaired in this line, but could be restored by addition of transportable auxins along with a normal organisation of actin (Maisch and Nick, 2007). Actin thus not only responds to changes in the cellular content of auxin, but actively participates in the establishment of the polarity that drives auxin transport. In the search for factors that regulate organisation and polarity of actin filaments, we cloned the tobacco actin-related protein 3 (ARP3) as a marker for sites of actin nucleation. This marker allowed to follow the behavior of actin-nucleation sites through patterned cell division (Maisch et al., 2009). The identification of Nicotiana tabacum actin-depolymerizing factor 2 (NtADF2) as auxin-signal mediator is the first attempt to link an actin-binding protein to auxin-driven actin reorganisation (Durst et al., 2013). NtADF2 controls the stability of fine actin filaments as precondition for functional auxin-dependent signalling. In addition, the stability of fine AF is involved in keeping the mentioned actin-auxin oscillator running. Future projects should extend our understanding towards the perception of auxin signals upstream of ADFs at the membrane.
Super-resolution microscopy in plant cell biology
We test applications of new microscopic methods such as PALM (Photoactivated Localization Microscopy) in living plant cells. We want to visualise different actin sub-populations and investigate functional compartmentalisation. In both cases we work with light-regulated switchable fluorescent molecules, so called pa-FPs (photoactivatable fluorescent proteins) that are fused to the actin marker life act.
In the meantime we were able to visualise a functional actin sub-population that forms a basket-like perinuclear structure. The resolution is in the range of 50 nm. The function of this sub-population is linked to nuclear positioning and migration (Durst et al., 2014).
In the future, we want to expand the application of these new photo-switchable FPs and establish a versatile vector system where - instead of a conventional FP - a pa-FP is used.
How is polarity generated de novo?
To study how cell polarity and axis are induced de novo, we established a regeneration system with protoplasts of tobacco BY-2 expressing fluorescently tagged cytoskeletal markers. We were able to generate quantitative data on the temporal patterns of regeneration stages, which allowed to establish and test models on the underlying cellular mechanisms (Zaban et al., 2013). It turned out that the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments. The new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules. Furthermore, we developed a system to integrate our regeneration approach into a microfluidics environment. This study suggests that the regenerating protoplast explores the geometry of its environment by sensing the local concentration of auxin extruded during regeneration.
How is the polarity of individual cells transformed into the supracellular pattern of a tissue?
A key event for patterning is cell division, because it defines symmetry, axis and orientation of the new cell wall. Whereas one pole can be inherited from the mother cell, there must be a de novo generation of a new cell pole at the site, where a new cross wall is laid down.
For this approach we follow the developmental history of individual cells over time from their first division to their differentiation into multi-cellular files. Therefore we have established long-term observation strategies.
Besides non-transformed BY-2 cells and actin marker lines we use other transgenic lines that allow the detection of auxin signalling (PIN, AUX) and intercellular auxin gradients (DR5).
On the other hand, we asked the question whether it is possible to manipulate the innate pattern of these files. In addition to inhibitor experiments, this can be done by caged auxin compounds that are used as a trigger for the defined release of auxin on the cellular level. We could demonstrate that the release of caged auxin within a single cell can manipulate the intracellular auxin level and the division pattern (Kusaka et al., 2009).
Applied science: molecular farming and microfluidics
Plant secondary metabolism generates numerous compounds of medical relevance whereas many of these compounds are produced only in specific cell types and require interaction and transport between different tissues. Therefore, it is a challenge to find alternative strategies in which the metabolic interaction of different cell types in a plant tissue can be technically mimicked.
We follow a biomimetic strategy that incorporates different cell types and transport events into molecular farming (patent pending). The basic idea is to assemble artificial “cages” containing genetically engineered suspension cells into a steady flow process line. The metabolism of upstream cells is genetically modified to yield a precursor product that is extruded. This product is then transported to the next cell type in the downstream cage which processes this precursor further and so on leading to the final compound. Our main goal is to engineer metabolic pathways leading to valuable compounds of medical relevance and to integrate metabolic modules into microfluidic devices for studying the interaction between different genes and gene products.
- Durst, Hedde PN, Brochhausen L, Nick P, Nienhaus GU, Maisch J (2014) Organization of perinuclear actin in live tobacco cells observed by PALM with optical sectioning. J Plant Physiol 141, 97-108 - pdf
- Dolch K, Danzer J, Kabbeck T, Bierer B, Erben J, Förster AH, Maisch J, Nick P, Kerzenmacher S, Gescher J (2014) Characterization of microbial current production as a function of microbe-electrode-interaction. Biores Technol 157, 284-292 - pdf
Durst S, Nick P, Maisch J (2013) Actin-Depolymerizing Factor 2 is Involved in Auxin Dependent Patterning, J Plant Physiol170, 1057-1066 - pdf
Maisch J, Fišerová J, Fischer L, Nick P (2009) Actin-related protein 3 labels actin-nucleating sites in tobacco BY-2 cells. J Exp Bot 60, 603-614 - pdf
Kusaka N, Maisch J, Nick P, Hayashi KI, Nozaki H (2009) Manipulation of Intercellular Auxin in a Single Cell by Light with Esterase-Resistant Caged Auxins. ChemBioChem 10, 2195-2202 - pdf
Maisch J, Nick P (2007) Actin is involved in auxin-dependent patterning. Plant Physiol 143, 1695-1704 - pdf