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+++ MITARBEITER - Projektbeschreibung (Markus Braun) +++

"Plant Gravity-Sensing and Gravity-Oriented Tip-growth"
Cellular Mechanisms and Molecular Components of
Gravitropic Signaling Pathways

PD Dr. Markus Braun

Topics of Research

My interests are in the area of plant gravitropic signaling pathways, with emphasis on the molecular and cellular aspects of gravisensing and graviresponses in unicellular model cell systems i.e. the tip-growing characeen rhizoids and protonemata

1. Cytoskeletal Organization in the Gravitropically Tip-growing cells

The highly dynamic actin and microtubule cytoskeletons form highly diverse MF-pattern in the apical, subapical and basal region of both tip-growing cell types, reflecting their specific features. In both cell types, two populations of thick and interconnected actin cables drive cytoplasmic streaming in the basal region. The actin cables fan out basal to the nucleus and form a dense meshwork of fine, axially oriented microfilament (MF) bundles in the apical and subapical region. In the apex, MTs are absent, whereas abundant actin MFs focus in a dense, spherical actin array that perfectly colocalizes with the apical ER aggregate in the center of the Spitzenkörper (apical body). Microinjection of fluorescently tagged tubulin showed that subapical MTs originate from the basal side of the nuclear envelope, indicating it“s MT-organizing function. MTs appear to flare out into the subapical zone, but, unlike actin filaments, end abruptly 35 - 40 µm basal from the tip. MTs are completely excluded from the apex of growing cells allowing unimpeded sedimentation of statoliths, a prerequisite for both gravisensing and the gravitropic responses. Terminating tip growth by mechanically manipulating the cells or by illuminating protonemata causes MTs to extend into the apical zone where they impair the gravity-directed statolith sedimentation.

2. Role of Actin and Calcium in Tip Growth

Polar growth relies on multiple actin-mediated functions of the tip-growth organizing Spitzenkörper, the delivery and coordinated incorporation of vesicles containing cell wall precursors, and the tip-high gradient of cytoplasmic calcium. The calcium gradient is maintained by apical calcium channels which I labeled with dihydro­pyridine (DHP). Disrupting the actin cytoskeleton with cytochalasin D reversibly altered the apical localization of the calcium channels, abolished the calcium gradient and stopped tip growth. The apical ER-aggregate appears to be involved in the regulation of the calcium gradient. It contains calcium-binding proteins like Caleosin which is proposed to function as a calcium sensor. The 23kD protein with a single EF hand might help to maintain the steepness of the calcium gradient by regulating ion pumps or channels in the ER-membranes. On the other hand, caleosin could also help to organize the ER-aggregate by regulating actin-associated proteins in a calcium dependent manner. A spectrin-like actin-binding protein (175 and 190 kD), which I detected exclusively in the area of the ER-aggregate in the Spitzenkörper center, seems to stabilize and organize the ER-aggregate by connecting the membranes to actin MFs. The spectrin labeling disappeared when tip growth stopped and the apical actin array and the ER-aggregate dispersed. Like in animal cells, the spectrin-like protein might also recruit a specific set of proteins to establish membrane microdomains that help to create the particular apical environ­ment for gravitropic tip growth.

3. Role of Actin in Susception and Gravity Sensing

Statoliths are transported and positioned by actin
Statoliths interact with the complexly organized actin MFs via myosins, which I immuno­fluorescently detected on the surface of statoliths. Experiments on clinostats and in microgravity (SpaceShuttle, sounding rockets: TEXUS-, MAXUS) revealed that the statoliths are kept in a dynamically stable position near the cell tip by two counteracting forces. In downward growing rhizoids, gravity acts on statoliths in acropetal direction (pulls statoliths into the tip) and actomyosin forces exactly compensate this force by acting net-basipetally. In upward growing protonemata, gravity acts basipetally (pulls the statoliths towards the cell base) and, therefore, the actomyosin forces act net-acropetally to compensate gravity and prevent the statoliths from settling towards the base of the cell. When statoliths were removed from the statolith region near the tip by centrifugation, statoliths were always retransported back to their original position.

Actin directs sedimenting statoliths to specific gravity perception sites
Upon horizontal positioning, gravity-directed sedimentation of statoliths was observed in rhizoids. In protonemata, however, the sedimenting statoliths were pushed into the tip by actin-mediated forces. Several experiments that I conducted in microgravity and on clinostats and centrifuges unambiguously demonstrated that the precise actin-controlled statolith positioning is neccessary to direct sedimenting statoliths to specific areas at the plasma membrane, the graviperception sites, where statolith sedimentation initiates the cellular response, the redirection of growth. By means of optical laser tweezers (optical trapping) or centrifugation, the sensitive membrane area was determined in rhizoids to be located 10-35 µm above the cell tip. In protonemata, the graviperception site was found to be at the very tip (0-10µm). Forcing statoliths to settle outside these areas did not lead to a gravitropic response. Thus, the actin cytoskeleton plays an important, but also a very different role in gravitropic sensing of both cell types.

4. Role of Actin and Calcium in Gravitropic Reorientation

Positive gravitropic response: differential flank growth
The positive gravitropic signal-transduction pathway is very short. The smooth bending down­ward is caused by differential growth of the opposite subapical cell flanks. Locally restricted lateral applications of CaCl2, calcium ionophores and GdCl3, a blocker of calcium channels, by means of a specially designed microinjection pipette, suggest that statolith sedimentation at the sensitive plasma-membrane area results in a local blockage of calcium channels. The result is a decrease in the concentration of cytoplasmic calcium and the inhibition of exocytosis. Recently, ratiometric calcium imaging that was conducted in Simon Gilroy“s Lab (Pennsylvania State University) confirmed our findings showing strongly reduced concentrations of cytoplasmic calcium at the lower cell flank at the beginning of the positive graviresponse. Work is in progress to elucidate the molecular nature of the membrane receptor and to clarify if membrane potential changes are involved in this process.

Negative gravitropic response: displacement of the growth center
In protonemata, the gravitropic signaling pathway is more complex. In rhizoids, I showed that the calcium gradient remains in a symmetrical position at the very tip during gravitropic bending. In contrast, in gravistimulated protonemata, there is a drastical redistribution of the calcium channel fluorescence and the calcium gradient towards the upper flank even before a change of the cell shape indicates the new direction of growth. This is strong evidence that statolith sedi­men­tation into the tip of protonemata causes a differential activation of calcium channels mainly on the upper flank of the apical dome. Accordingly, the calcium gradient moves towards the upper flank and along this moving gradient actin-binding proteins could be activated that help to displace the whole Spitzenkörper and, thus, the center of growth to the upper flank. I found myosin VIII specifically located in the apical plasma membrane that represents a good candidate for this function. Direct proof for the actual displacement of the Spitzenkörper comes from a spectrin labeling that was used as a marker to indicate the position of the Spitzenkörper center. In rhizoids, the center always remained in a symmetrical position close to the very tip during gavi­tropic bending, whereas in protonemata, the labeling indicated a drastical displace­ment of the center towards the upper flank at the beginning of the gravitropic reorientation.

It is concluded that in rhizoids, the Spitzenkörper and the center of growth is more firmly anchored at the cell tip by cytoskeletal forces than in protonemata. With centrifugation and optical tweezers, it was possible to overcome the original forces and to displace the Spitzen­körper in rhizoids by mechanically pushing the statoliths into the cell tip. This manipulation resulted in a protonema-like response in rhizoids.

5. Gravitropic Tip Growth Requires a Complex Cytoskeletal Organization and a Concerted Action of Multiple Actin Associated Proteins

An apical actin MF-arrangement and a Spitzenkörper as complexly organized as in characean rhizoids and protonemata has not been found in any other tip-growing cell type. Especially the center of the Spitzenkörper with its dense actin array and the aggregation of ER is a unique, fascinating compartment with diverse functions reflected by a number of proteins that I localized in this area. As was mentioned above, the oleosin-related caleosin in the ER-membranes might serve as a sensor for regulating the tip-high gradient of cytoplasmic calcium. Interestingly, the center of the Spitzenkörper appears also to function as an amazingly clearly defined apical polymerization site for the apical actin MFs. Recent cytochalasin experiments and subsequent actin labeling showed that after complete depolymerization reorganization of the apical actin MFs starts with the formation of a new dense actin array at the most apical plasma membrane area. The actin array grows, becomes spherical again and accumulates ER-membranes. As soon as MFs emerge asterlike from the sphere, it moves basipetally and, eventually, takes its original position in the center of the apical dome and tip growth resumes.

The localization of ADF (actin depolymerizing factor) and profilins in the center of the Spitzenkörper is a further strong argument for its role as an actin-polymerization site since these proteins indicate high actin turn-over and reorganization.

In cooperation with Klaus Palme“s Group (Max Delbrück Laboratorium in der Max-Planck-Gesellschaft, Köln), we study the role of Rho-GTPases as molecular switches for polar growth in root hairs (GFP-AtRop4, AtRop6) and we started to analyse the function of Rho-GTPases in gravitropic tip growth.

6. Molecular Components of Gravitropic Signaling Pathways

Currently, we focus on molecular approaches to characterize and identify actin-binding and other tip-growth related proteins in both genetically identical and morphologically very similar cell types. Myosins, which are key players for the actin-mediated processes of tip growth and negativ and positiv graviorientation, are genetically analysed and immuncytochemically localized in both cell types. Differential-display analysis (involving RNA-extraction, RT-PCR, capillary electro­phoresis, cloning and sequenzing etc.) is in progress that will help us to identify differentially expressed genes representing molecular components and possible key elements (molecular switches) for negative and positive gravitropic signaling pathways. It will then be tested, if the findings from the unicellular model systems can help to uncover the molecular mechanisms of gravity sensing and gravitropic responses in other unicellular systems and also in higher plants.

Experiences and Methods

- Transmission und scanning electron microscopy, X-ray microanalysis,
- High-pressure freeze fixation, rapid-freeze fixation, freeze substitution,
- Immunogold localization
- Confocal laserscannig microscopy, immunfluorescence microscopy
- Laser micromanipulation (optical tweezers)
- Microinjection, ratiometric fluorescence calcium imaging, GFP-imaging
- Protein biochemistry (extraction, SDS-PAGE, western-, immunoblotting, overlay assays)
- Video microscopy and digital image analysis, statistical analysis
- Molecular biology (GFP, PCR, RT-PCR, Klonierung, particle gun, RFLP-gene mapping)

Experience in manned space flight (Space Shuttle missions) and sounding rocket
campagnes (TEXUS, MAXUS)

© Institut für Molekulare Physiologie und Biotechnologie der Pflanzen IMBIO, AG Gravitationbiologie  ( © Fabian A. Paul )