Research


 

A brief description of some of our current research topics:
 

Spotlighted brains: Optogenetic activation and silencing of neurons
Optogenetics is revolutionizing cell biology and neuroscience research by allowing precise biochemical control of neuronal activity through light-activated channels. Light-induced ion transporters have been used extensively for cellular activation, and now light-gated inhibitory channels have been discovered. These represent a key new tool to elucidate the molecular mechanisms underlying neurological and neuropsychiatric disorders..
Reference: Kianianmomeni, A. & Hallmann, A. (2015). Trends Biochem. Sci. 40, 624-627.

 
Algal photoreceptors: in vivo functions and potential applications
Many algae, particularly microalgae, possess a sophisticated light-sensing system including photoreceptors and light-modulated signaling pathways to sense environmental information and secure the survival in a rapidly changing environment. Over the last couple of years, the multifaceted world of algal photobiology has enriched our understanding of the light absorption mechanisms and in vivo function of photoreceptors. Moreover, specific light-sensitive modules have already paved the way for the development of optogenetic tools to generate light switches for precise and spatial control of signaling pathways in individual cells and even in complex biological systems.
Reference: Kianianmomeni, A. & Hallmann, A. (2014). Planta 239, 1-26.
 
The genome of Volvox, the “fierce roller”: Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri
The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-megabase genome of V. carteri and compared its ~14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal–specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.
Reference: Prochnik, S. E., Umen, J., Nedelcu, A., Hallmann, A. et al. (2010). Science 329, 223-226.
 
There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator
Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult conguration; this process is called "inversion." There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out? We investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri. Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator.
Reference: Höhn, S. & Hallmann, A. (2011). BMC Biol 9, 89.
 

A gender-specific retinoblastoma-related protein implies a role for the retinoblastoma protein family in sexual development
The green alga Volvox carteri is one of the simplest multicellular organisms with only two cell types, somatic and reproductive, making it suitable as a model for studying cell division, multicellularity, and cellular differentiation. We cloned and characterized the RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) from the green alga Volvox carteri. Likewise, other key elements of the retinoblastoma tumor suppressor pathway like E2F1 and DP1 have been identified in Volvox carteri. RBR1 expression increases substantially during embryogenesis and in response to the sex-inducer glycoprotein, but it decreases significantly under heat stress. While RBR1 is expressed in gonidia (asexual reproductive cells) and embryos, the largest proportion of RBR1 mRNA is found in parental somatic cells. The presence of 4 splice variants and 15 potential cyclin-dependent kinase phosphorylation sites suggests that RBR1 is subject to control at the posttranscriptional and posttranslational levels. Surprisingly, RBR1 is a gender-specific gene, mapping exclusively to the female mating-type locus. A procedure for stable nuclear transformation of males was established to generate RBR1-expressing males. These transformants exhibit enlarged reproductive cells, altered growth characteristics, and a prolonged embryogenesis. The results suggest that a functionally related analog of RBR1 exists in males. The reason for the divergent evolution of RBRs in females and males appears to be based on sexual development: males and females respond to the same sex-inducer with different cleavage programs and substantial differences in cellular differentiation. Thus, the gender-specific presence of RBR1 provides evidence for an additional, novel role for retinoblastoma family proteins in sexual development.
References: Kianianmomeni, A., Nematollahi, G. & Hallmann, A. (2008). Plant Cell 20, 2399-2419. __Hallmann, A. (2009). Commun. Integr. Biol. 2, 396-399. __Hallmann, A. (2009). Commun. Integr. Biol. 2, 538-544.

 
How 5000 independent rowers coordinate their strokes in order to row into the sunlight: Phototaxis in the multicellular green alga Volvox
The evolution of multicellular motile organisms from unicellular ancestors required the utilization of previously evolved tactic behavior in a multicellular context. Volvocine green algae are uniquely suited for studying tactic responses during the transition to multicellularity because they range in complexity from unicellular to multicellular genera. Phototactic responses are essential for these flagellates because they need to orientate themselves to receive sufficient light for photosynthesis, but how does a multicellular organism accomplish phototaxis without any known direct communication among cells? Several aspects of the photoresponse have previously been analyzed in volvocine algae, particularly in the unicellular alga Chlamydomonas. Recently, we analyzed the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta). In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. Moreover, positive phototaxis is facilitated by gravitational forces. Under our conditions, V. rousseletii spheroids showed no negative phototaxis. On the basis of our results, we developed a mechanistic model that predicts the phototactic behavior in V. rousseletii. The model involves photoresponses, periodically changing light conditions, morphological polarity, rotation of the spheroid, two modes of flagellar beating, and the impact of gravity. Our results also indicate how recently evolved multicellular organisms adapted the phototactic capabilities of their unicellular ancestors to multicellular life.
Reference: Ueki, N., Matsunaga, S., Inouye, I. & Hallmann, A. (2010). BMC Biol. 8, 103.
 
Light-gated ion channels that control photomovement responses: The Channelrhodopsins
Channelrhodopsins are light-gated ion channels involved in the photoresponses of microalgae. Here we describe the characterization of two channelrhodopsins, VChR1 and VChR2, from the multicellular green alga Volvox carteri. Both are encoded by nuclear single copy genes and are highly expressed in the small biflagellated somatic cells but not in the asexual reproductive cells (gonidia). Expression of both VChRs increases after cell cleavage and peaks after completion of embryogenesis when the biosynthesis of the extracellular matrix begins. Likewise, expression of both transcripts increases after addition of the sex-inducer protein, but VChR2 is induced much more than VChR1. The expression of VChR1 is specifically promoted by extended dark periods, and heat stress reduces predominantly VChR1 expression. Expression of both VChRs increased under low light conditions, whereas cold stress and wounding reduced expression. Both VChRs were spectroscopically studied in their purified recombinant forms. VChR2 is similar to the ChR2 counterpart from Chlamydomonas reinhardtii with respect to its absorption maximum (460 nm) and photocycle dynamics. In contrast, VChR1 absorbs maximally at 540 nm at low pH (D540), shifting to 500 nm at high pH (D500). Flash photolysis experiments showed that after light excitation, the D540 dark state bleaches and at least two photoproducts, P600 and P500, are sequentially populated during the photocycle. We hypothesize that VChR2 is a general photoreceptor, which is responsible for the avoidance of blue light and might play a key role in sexual development, whereas VChR1 is the main phototaxis photoreceptor under vegetative conditions, as it is more specifically adapted to environmental conditions and the developmental stages of Volvox.
Reference: Kianianmomeni, A., Stehfest, K., Nematollahi, G., Hegemann, P. & Hallmann, A. (2009). Plant Physiol. 151, 347-366.
 
VCRPs, small cysteine-rich proteins that might be involved in extracellular signaling
The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10 (-16) M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism’s environment to a reproductive cell must involve the ECM. A small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculatedto be an extracellular second messenger from somatic cells to reproductive cells. There is also a related protein, VCRP2, which exhibits 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.
In the future, we want to investigated this signal transduction in more detail to learn more about the corresponding molecular mechanisms.
References: Hallmann, A. (2008). Plant Signal. Behav. 3, 124-127. __Hallmann, A. (2007). Planta 226, 719-727.
 
The Chlamydomonas genome reveals the evolution of key animal and plant functions
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella.
Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
Reference: Merchant, S. S. et al. (2007). Science 318, 245-250.
 
Stable nuclear transformation of volvocine algae
Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. For a detailed analysis of  this molecular evolution, transformation techniques are required, which allow for genetic manipulation of these algae. Until recently, transformation procedures have only been established for the volvocine algae Chlamydomonas reinhardtii and Volvox carteri. Now we achieved the stable nuclear transformation of Gonium pectorale. Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. Stable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of ~30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays. Conclusions: Flanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox.
Reference: Lerche, K. & Hallmann, A. (2009). BMC Biotechnol. 9, 64.
 
Algal transgenics and biotechnology
Transgenesis in algae is a complex and fast-growing technology. Selectable marker genes, promoters, reporter genes, transformation techniques, and other genetic tools and methods are already available for various species and currently ~25 species are accessible to genetic transformation. Fortunately, large-scale sequencing projects are also planned, in progress, or completed for several of these species; the most advanced genome projects are those for the red alga Cyanidioschyzon merolae, the diatom Thalassiosira pseudonana, and the three green algae Chlamydomonas reinhardtii, Volvox carteri and Ostreococcus tauri. The vast amount of genomic and EST data coming from these and a number of other algae has the potential to dramatically enlarge not only the algae’s molecular toolbox. A powerful driving force in algal transgenics is the prospect of using genetically modified algae as bioreactors. In general, today’s non-transgenic, commercial algal biotechnology produces food additives, cosmetics, animal feed additives, pigments, polysaccharides, fatty acids, and biomass. But recent progress in algal transgenics promises a much broader field of application: molecular farming, the production of proteins or metabolites that are valuable to medicine or industry, seems to be feasible with transgenic algal systems. Indeed, the ability of transgenic algae to produce recombinant antibodies, vaccines, insecticidal proteins, or bio-hydrogen has already been demonstrated.
Genetic modifications that enhance physiological properties of algal strains and optimization of algal production systems should further improve the potential of this auspicious technology in the future.
Reference: Hallmann, A. (2007). Transgenic Plant J. 1, 81-98.
 

Quantitative analysis of cell-type specific gene expression
The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there is an ongoing genome project in Volvox carteri and a completed genome project in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluated the potential of real-time RT-PCR for quantifying gene transcript levels. We analyzed a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contained previously known genes with unknown localization of cellular expression, 28 novel genes which were described for the first time, and a few known, cell-type specific genes as a control. The respective gene products were, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provided expression data for all these genes. The results showed that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox. Our expression data also provided a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.
In the future, this approach will be extended to a much larger number of genes and to developmental or metabolic mutants.
Reference: Nematollahi, G., Kianianmomeni, A. & Hallmann, A. (2006). BMC Genomics 7, 321.

 

Translational control of a key gene, regA, controlling cell differentiation
The complete division of labour between the reproductive and somatic cells of the green alga Volvox carteri is controlled by three types of genes. One of these is the regA gene, which controls terminal differentiation of the somatic cells. We examined translational control elements located in the 5' UTR of regA, particularly the eight upstream start codons (AUGs) that have to be bypassed by the translation machinery before regA can be translated. The results of our systematic mutational, structural and functional analysis of the 5' UTR led us to conclude that a ribosome-shunting mechanism - rather than leaky scanning, ribosomal reinitiation, or internal ribosome entry site (IRES)-mediated initiation - controls the translation of regA mRNA. This mechanism, which involves dissociation of the 40S initiation complex from the message, followed by reattachment downstream, in order to bypass a secondary structure block in the mRNA, was validated by deleting the predicted 'landing site' (which prevented regA expression) and inserting a stable 64 nucleotide hairpin just upstream of this site (which did not prevent regA expression). This is the first report suggesting that translation of an mRNA in a green eukaryote is controlled by ribosome shunting.
In the future, we want to investigate whether other key genes in development are also controlled by ribosome shunting.
Reference: Babinger, K., Hallmann, A. & Schmitt, R. (2006). Development 133, 4045-4051.

 
 

 

 


Copyright © 2015 by Prof. Dr. Armin Hallmann, University of Bielefeld, Germany