- Email: [email protected]
Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular morphogenesis
Review
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular * morphogenesis Salvatore Bozzaro1, Paul R. Fisher2, William Loomis3, Peter Satir4 and Jeffrey E. Segall4 1
Clinical and Biological Sciences, University of Torino, Orbassano 10043, Italy Department of Microbiology, La Trobe University, Victoria 3086, Australia 3 Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA 4 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2
Beginning in 1960 and continuing to this day, Guenther Gerisch’s work on the social ameba Dictyostelium discoideum has helped to make it the model organism of choice for studies of cellular activities that depend upon the actomyosin cytoskeleton. Gerisch has brought insight and quantitative rigor to cell biology by developing novel assays and by applying advanced genetic, biochemical and microscopic techniques to topics as varied as cell–cell adhesion, chemotaxis, motility, endocytosis and cytokinesis.
biologist Wolfhard Weidel, who studied mainly phage and bacteria but who was also looking for a suitable person to study D. discoideum. This was at a time when
As a high-school student in Dresden in the late 1940s, Guenther Gerisch (Figure 1) first learned of and was fascinated by the organism that would form the basis of his scientific career. In the 1930s, Arthur Arndt had made a movie of Dictyostelium discoideum development that showed how these eukaryotic cells aggregate and form fruiting bodies, which inspired biologists and biochemists to study them further. D. discoideum cells live in soil as single amebae, feeding on bacteria (Figure 2). However, upon starvation, these amebae begin communicating with each other, sending signals that enable them to come together, or aggregate, by chemotaxis and adhesion. The multicellular aggregate resembles a developing tissue and differentiates further, forming a migrating ‘slug’ that then converts itself into a spore-containing sorus that sits upon a stalk made up of tightly packed, vacuolated cells. Thus, this species provides a dramatic example of the conversion of single cells into a multicellular structure and it is particularly amenable for genetic study because neither aggregation nor fruiting-body formation is essential for the survival of D. discoideum amebae. As a graduate student at the Max-Planck-Institut fu¨r Biologie at Tuebingen (http://www.tuebingen.mpg.de/), Gerisch worked in the laboratory of the molecular Corresponding author: Jeffrey E. Segall ([email protected]). This article is part of the Pioneers series in Trends in Cell Biology. Pioneers articles feature researchers, experiments and concepts that forged the way to modern cell biology. *
Figure 1. Guenther Gerisch in the library of his department at the Max-PlanckInstitut fu¨r Biochemie (http://www.biochem.mpg.de/) during a retirement party for Monika Westphal (a long-time collaborator). Image provided by Mary Ecke.
www.sciencedirect.com 0962-8924/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.09.006
586
Review
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Figure 2. The life cycle of Dictyostelium discoideum. (i) Immunofluorescence of LIM delE–GFP (green fluorescent protein) dynamics. Reprinted, with permission, from Ref. [11]. (ii) GFP–actin and lysotracker-red-labeled cell. Reprinted, with permission, from Ref. [12]. q (2001) Wiley-Liss, a subsidiary of John Wiley & Sons. (iii) Scanning electron microscopy (SEM) image of aggregating cells in a stream. (iv) A small aggregate. (v) An aggregation field. (vi) Migrating slugs. (vii) A sorus containing spores.
many biologists were looking for simple, often microbial, models that would enable them to apply the enormously successful approaches used in bacterial and phage genetics to eukaryotic cell and developmental biology. Working alone on the subject in Weidel’s laboratory, Gerisch discovered conditions for the precise and reproducible initiation of development in D. discoideum. He found that the first 8 h of differentiation, during which the cells become aggregation competent, could occur in a shaken suspension of washed amebae in simple buffers. Although later development would occur only on a surface at an air–water interface, the ability to study early differentiation events on cells in suspension made their biochemical and physiological analysis much easier. During these studies, Gerisch was struck by two features of D. discoideum development that were to form the basis of much of his future work: (i) during aggregation, cells surged towards aggregating centers in a periodic manner that generated self-propagating waves and spirals; and (ii) as the cells started moving together, they became mutually adhesive and stuck together strongly.
development, they identified two antigenically distinct mechanisms for cell–cell adhesion. The first, which was termed ‘contact-sites A’, arose during early differentiation and was unaffected by EDTA (because it was Ca2C independent), whereas a second mechanism (‘contactsites B’) was already present in vegetative cells and was blocked by EDTA. These adhesion studies were influential in generating a paradigm shift from a view in which cell adhesion was the sum of nonspecific interactions between two membranes to a view in which adhesion could be divided into specific interactions between membrane constituents. Furthermore, to this day, the Gerisch adhesion assay forms the basis for most adhesion assays of mammalian cells. After many years of continuously refining the approach, Gerisch and colleagues were able to purify and characterize the peripheral plasma-membrane protein that mediates the EDTA-stable adhesion: gp80. This was one of the first cell–cell adhesion proteins to be identified in any organism, and the antibody-based approach that Gerisch used was also applied successfully to the cell-adhesion molecules (CAMs) of other organisms.
The mechanism of cell–cell adhesion Gerisch focused initially on the acquisition of cell–cell contacts during differentiation and began a series of studies of the plasma membrane and its constituents to identify these contacts. These studies contributed to the acceptance of the plasma membrane as being a valid cellular structure with defined molecular constituents. In an article published in 1959 [1], he showed that cell–cell adhesion was sensitive to high levels of EDTA during the first 6 h of development but, thereafter, became EDTA stable. The initial adhesion assays depended on microscopic observations of clumps of cells but, to obtain higher quantitative resolution, Gerisch and Hartmut Beug (a student in his laboratory) used suspensions of cells in cuvettes and measured light scattering [2]. They produced antibodies against plasma-membrane proteins and devised strategies using monovalent antibodies with the appropriate controls to show specificity at inhibiting cell–cell adhesion [3,4]. By absorbing the antibodies with membrane fractions from cells at different stages of
Biochemistry of the relay and transduction of the chemotactic signal As the adhesion studies matured, Gerisch spent more time studying the oscillations and spirals that he had seen during his studies of aggregation. He predicted that such oscillations implied fluctuations in the rate of energy use, leading to periodic changes in the energy state of the cells. In collaboration with Benno Hess, he tested this prediction, using difference spectra to measure NAD and NADP oxidation levels in the cuvettes in which suspended cells could develop. There were reproducible oscillations but they were relatively small. These measurements were based on subtracting a reference wavelength, and Hess had enough recorders to measure the reference-wavelength absorbance and the difference value. This enabled Gerisch to discover that there were huge oscillations in the reference-wavelength absorbance, which he found were based on changes in light scattering due to changes in cell shape [5]. This inspired a series of experiments demonstrating that the oscillations in light scattering were
www.sciencedirect.com
Review
TRENDS in Cell Biology
43"
K
57"
L
Vol.14 No.10 October 2004
63"
M
587
71"
N
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O
Figure 3. Chemotactic responses of a Dictyostelium discoideum cell to a micropipette filled with cyclic AMP (cAMP). The cell is moving towards the micropipette at the top of the image in K. In L, the pipette has been moved to the lower left and the cell reorients by retracting its protrusion at the top of the field and extending a new one towards the pipette. Time (min) shown in upper right of images. Scale bar, 20 mm. Reprinted, with permission, from Ref. [13]. q (1986) Company of Biologists.
synchronized with spontaneous oscillations in cyclic AMP (cAMP) levels. Several years earlier, cAMP had been identified as being a chemoattractant for D. discoideum by Theo Konijn and John Bonner [6]. Gerisch showed that a pulse of secreted cAMP not only induced chemotaxis in a neighboring cell but also induced this cell to secrete its own burst after a short time period, which then caused chemoattraction and cAMP production in another nearby cell. Thus, the chemoattractant signal was identical to the signal relayed in the field of aggregating cells. This concept also explained how chemotaxis could assemble cells over distances (up to 0.5 cm) that were far too large to enable sensing of a simple cAMP gradient and, thus, provided a mechanism that coupled oscillations to chemotaxis for efficient aggregation. This experimental system, in which the periodic oscillations essential for aggregation could be reproducibly generated in dense cell suspensions, enabled Gerisch to explore biochemical signaling pathways downstream of cAMP stimulation, including those involving intracellular pH changes, Ca2C fluxes, cAMP synthesis, cyclic GMP (cGMP) production and cytoskeletal regulation. With Dieter Malchow and two students in his laboratory (Werner Roos and Ursula Wick), a series of ingenious experiments overcame the difficulties posed by high phosphodiesterase activity and demonstrated both the binding of cAMP to the cell surface and the cAMPstimulated synthesis and secretion of new cAMP (a process known as cAMP relay) [7]. The role of the actomyosin cytoskeleton in cellular motility The dramatic oscillations in light scattering in response to cAMP reflect cytoskeletal rearrangements that are caused by the chemotactic response that enables D. discoideum cells to aggregate. Beginning in the 1980s and continuing to this day, Gerisch has studied the cytoskeletal changes that occur not only in the chemotactic motility of ameboid cells but also in phagocytosis, pinocytosis and mitosis, using D. discoideum as a model and combining its emerging genetics with high-resolution microscopy. An important microscopic assay for these studies grew out of an attempt with Dieter Huelser to record membranepotential changes in response to stimulation with chemoattractant. Although the membrane properties of the cells made the original goal of making membrane-potential measurements impossible, a valuable result was the www.sciencedirect.com
discovery that a micropipette filled with cAMP could be used to stimulate cells locally, enabling the direct recording of shape changes during the chemotaxis of single amebae (Figure 3). One of the early surprises that came from the genetic studies by Angelika Noegel and Michael Scheicher [8] was the apparent redundancy of components. Genetic ablation of what were thought to be major cytoskeletal proteins, such as a actinin, had only minor effects on cell movement and chemotaxis. This was surprising because it was expected that mutants lacking these proteins would show more-dramatic phenotypes. Today, the concept of redundancy is pervasive, with knockout mouse studies often finding minimal phenotypes. But the meaning of redundancy is still debated, with many researchers suggesting that, under critical conditions, no two proteins will be totally redundant. Recently, Gerisch has returned to one of his early passions – meticulous imaging studies – embarking with Annette Muller-Taubenberger on a detailed examination of the dynamics and interactions between cell-substratum adhesion and proteins such as the Arp2/3 complex and talin [9]. In addition to the elegant light-microscopic analysis of green fluorescent protein (GFP) fusion proteins, Gerisch and Igor Weber have joined forces with Wolfgang Baumeister and Ohad Medalia to show, by way of the actin cytoskeleton example, that cryoelectron tomography can be used to study the 3D topology of cellular macromolecules in situ at a resolution of 5–6 nm [10] (Figure 4). In combination with the many D. discoideum mutants lacking specific cytoskeletal proteins, this technique promises insights of unparalleled depth and detail into the cellular functions of the cytoskeleton. Still working at the cutting edge with his characteristic commitment to precision and rigor, Gerisch continues to advance our understanding of a range of biological processes, including cell motility, endocytosis, cell division and adhesion. Concluding remarks The legacy of influential scientists extends beyond the work they, themselves, have done to the impact that they have had on the careers of others in their field and the research carried out by those people – their scientific offspring, as it were. During nearly half a century of sustained effort, Guenther Gerisch has inspired colleagues with the development of new methods that enable
588
Review
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Gerisch’s own laboratory has been enormous. Gerisch’s decision to study the role of the actomyosin cytoskeleton in chemotactic motility has, thus, resulted in D. discoideum becoming the model of choice and easily the best-understood system in this area. The lessons learned from D. discoideum have contributed to the understanding of such phenomena as leukocyte chemotaxis towards wound and inflammation sites, tumor cell motility and neuronal targeting in the central nervous system. Acknowledgements We thank the many colleagues of Guenther Gerisch who contributed helpful comments and we apologize for not contacting more of Guenther’s colleagues so that they could also have contributed.
References
Figure 4. Visualization by cryoelectron tomography of the Dictyostelium discoideum actin network, membranes and cytoplasmic macromolecular complexes. A volume of 815 ! 970 ! 97 nm3 was subjected to surface rendering. Colors were subjectively attributed to linear elements to mark the actin filaments (red), other macromolecular complexes (mostly ribosomes) (green) and membranes (blue). Reprinted, with permission, from Ref. [10]. q (2002) American Association for the Advancement of Science (http://www.sciencemag.org/).
imaginative and careful experiments essential for identifying the specific contributions of individual proteins to cellular processes, combined with a broad interest in how cells work as integrated systems. Many of the D. discoideum research groups around the world, from places as far flung as Australia to the USA and various European countries, are led by people who passed through the Gerisch Cell Biology Department in Martinsried, either as PhD students or as postdoctoral researchers. Particularly in the area of nonmuscle cell motility and chemotaxis, the collective impact of their work and the ongoing work in
1 Gerisch, G. (1959) Ein submerskulturver fahren fur entwicklungsphysiologische untersuchungen an Dictyostelium discoideum. Naturwissenschaften 46, 654–656 2 Beug, H. and Gerisch, G. (1972) A micromethod for routine measurement of cell agglutination and dissociation. J. Immunol. Methods 2, 49–57 3 Beug, H. et al. (1971) Cell dissociation: univalent antibodies as a possible alternative to proteolytic enzymes. Science 173, 742–743 4 Muller, K. and Gerisch, G. (1978) A specific glycoprotein as the target site of adhesion blocking Fab in aggregating Dictyostelium cells. Nature 274, 445–449 5 Gerisch, G. and Hess, B. (1974) Cyclic-AMP-controlled oscillations in suspended Dictyostelium cells: their relation to morphogenetic cell interactions. Proc. Natl. Acad. Sci. U. S. A. 71, 2118–2122 6 Konijn, T. et al. (1968) Cyclic AMP: a naturally occurring acrasin in the cellular slime molds. Am. Nat. 102, 225–233 7 Gerisch, G. et al. (1979) Oscillations of cyclic nucleotide concentrations in relation to the excitability of Dictyostelium cells. J. Exp. Biol. 81, 33–47 8 Gerisch, G. et al. (1991) Genetic alteration of proteins in actin-based motility systems. Annu. Rev. Physiol. 53, 607–628 9 Gerisch, G. and Muller-Taubenberger, A. (2003) GFP-fusion proteins as fluorescent reporters to study organelle and cytoskeleton dynamics in chemotaxis and phagocytosis. Methods Enzymol. 361, 320–337 10 Medalia, O. et al. (2002) Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science 298, 1209–1213 11 Bretschneider, T. et al. (2004) Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr. Biol. 14, 1–10 12 Insall, R. et al. (2001) Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis. Cell Motil. Cytoskeleton 50, 115–128 13 Claviez, M. et al. (1986) Cytoskeletons from a mutant of Dictyostelium discoideum with flattened cells. J. Cell Sci. 86, 69–82
Trends in Cell Biology: new impact factor! A big thank you from the editorial team to all our readers. Thanks to you, the 2003 ISI impact factor for Trends in Cell Biology is 19.6. Trends in Cell Biology continues to be one of the most high-profile journals in which to publish! Cristina Pelizon Editor www.sciencedirect.com
Peter Chapman Assistant Editor
Helen Gannon Editorial Coordinator
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular * morphogenesis Salvatore Bozzaro1, Paul R. Fisher2, William Loomis3, Peter Satir4 and Jeffrey E. Segall4 1
Clinical and Biological Sciences, University of Torino, Orbassano 10043, Italy Department of Microbiology, La Trobe University, Victoria 3086, Australia 3 Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA 4 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2
Beginning in 1960 and continuing to this day, Guenther Gerisch’s work on the social ameba Dictyostelium discoideum has helped to make it the model organism of choice for studies of cellular activities that depend upon the actomyosin cytoskeleton. Gerisch has brought insight and quantitative rigor to cell biology by developing novel assays and by applying advanced genetic, biochemical and microscopic techniques to topics as varied as cell–cell adhesion, chemotaxis, motility, endocytosis and cytokinesis.
biologist Wolfhard Weidel, who studied mainly phage and bacteria but who was also looking for a suitable person to study D. discoideum. This was at a time when
As a high-school student in Dresden in the late 1940s, Guenther Gerisch (Figure 1) first learned of and was fascinated by the organism that would form the basis of his scientific career. In the 1930s, Arthur Arndt had made a movie of Dictyostelium discoideum development that showed how these eukaryotic cells aggregate and form fruiting bodies, which inspired biologists and biochemists to study them further. D. discoideum cells live in soil as single amebae, feeding on bacteria (Figure 2). However, upon starvation, these amebae begin communicating with each other, sending signals that enable them to come together, or aggregate, by chemotaxis and adhesion. The multicellular aggregate resembles a developing tissue and differentiates further, forming a migrating ‘slug’ that then converts itself into a spore-containing sorus that sits upon a stalk made up of tightly packed, vacuolated cells. Thus, this species provides a dramatic example of the conversion of single cells into a multicellular structure and it is particularly amenable for genetic study because neither aggregation nor fruiting-body formation is essential for the survival of D. discoideum amebae. As a graduate student at the Max-Planck-Institut fu¨r Biologie at Tuebingen (http://www.tuebingen.mpg.de/), Gerisch worked in the laboratory of the molecular Corresponding author: Jeffrey E. Segall ([email protected]). This article is part of the Pioneers series in Trends in Cell Biology. Pioneers articles feature researchers, experiments and concepts that forged the way to modern cell biology. *
Figure 1. Guenther Gerisch in the library of his department at the Max-PlanckInstitut fu¨r Biochemie (http://www.biochem.mpg.de/) during a retirement party for Monika Westphal (a long-time collaborator). Image provided by Mary Ecke.
www.sciencedirect.com 0962-8924/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.09.006
586
Review
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Figure 2. The life cycle of Dictyostelium discoideum. (i) Immunofluorescence of LIM delE–GFP (green fluorescent protein) dynamics. Reprinted, with permission, from Ref. [11]. (ii) GFP–actin and lysotracker-red-labeled cell. Reprinted, with permission, from Ref. [12]. q (2001) Wiley-Liss, a subsidiary of John Wiley & Sons. (iii) Scanning electron microscopy (SEM) image of aggregating cells in a stream. (iv) A small aggregate. (v) An aggregation field. (vi) Migrating slugs. (vii) A sorus containing spores.
many biologists were looking for simple, often microbial, models that would enable them to apply the enormously successful approaches used in bacterial and phage genetics to eukaryotic cell and developmental biology. Working alone on the subject in Weidel’s laboratory, Gerisch discovered conditions for the precise and reproducible initiation of development in D. discoideum. He found that the first 8 h of differentiation, during which the cells become aggregation competent, could occur in a shaken suspension of washed amebae in simple buffers. Although later development would occur only on a surface at an air–water interface, the ability to study early differentiation events on cells in suspension made their biochemical and physiological analysis much easier. During these studies, Gerisch was struck by two features of D. discoideum development that were to form the basis of much of his future work: (i) during aggregation, cells surged towards aggregating centers in a periodic manner that generated self-propagating waves and spirals; and (ii) as the cells started moving together, they became mutually adhesive and stuck together strongly.
development, they identified two antigenically distinct mechanisms for cell–cell adhesion. The first, which was termed ‘contact-sites A’, arose during early differentiation and was unaffected by EDTA (because it was Ca2C independent), whereas a second mechanism (‘contactsites B’) was already present in vegetative cells and was blocked by EDTA. These adhesion studies were influential in generating a paradigm shift from a view in which cell adhesion was the sum of nonspecific interactions between two membranes to a view in which adhesion could be divided into specific interactions between membrane constituents. Furthermore, to this day, the Gerisch adhesion assay forms the basis for most adhesion assays of mammalian cells. After many years of continuously refining the approach, Gerisch and colleagues were able to purify and characterize the peripheral plasma-membrane protein that mediates the EDTA-stable adhesion: gp80. This was one of the first cell–cell adhesion proteins to be identified in any organism, and the antibody-based approach that Gerisch used was also applied successfully to the cell-adhesion molecules (CAMs) of other organisms.
The mechanism of cell–cell adhesion Gerisch focused initially on the acquisition of cell–cell contacts during differentiation and began a series of studies of the plasma membrane and its constituents to identify these contacts. These studies contributed to the acceptance of the plasma membrane as being a valid cellular structure with defined molecular constituents. In an article published in 1959 [1], he showed that cell–cell adhesion was sensitive to high levels of EDTA during the first 6 h of development but, thereafter, became EDTA stable. The initial adhesion assays depended on microscopic observations of clumps of cells but, to obtain higher quantitative resolution, Gerisch and Hartmut Beug (a student in his laboratory) used suspensions of cells in cuvettes and measured light scattering [2]. They produced antibodies against plasma-membrane proteins and devised strategies using monovalent antibodies with the appropriate controls to show specificity at inhibiting cell–cell adhesion [3,4]. By absorbing the antibodies with membrane fractions from cells at different stages of
Biochemistry of the relay and transduction of the chemotactic signal As the adhesion studies matured, Gerisch spent more time studying the oscillations and spirals that he had seen during his studies of aggregation. He predicted that such oscillations implied fluctuations in the rate of energy use, leading to periodic changes in the energy state of the cells. In collaboration with Benno Hess, he tested this prediction, using difference spectra to measure NAD and NADP oxidation levels in the cuvettes in which suspended cells could develop. There were reproducible oscillations but they were relatively small. These measurements were based on subtracting a reference wavelength, and Hess had enough recorders to measure the reference-wavelength absorbance and the difference value. This enabled Gerisch to discover that there were huge oscillations in the reference-wavelength absorbance, which he found were based on changes in light scattering due to changes in cell shape [5]. This inspired a series of experiments demonstrating that the oscillations in light scattering were
www.sciencedirect.com
Review
TRENDS in Cell Biology
43"
K
57"
L
Vol.14 No.10 October 2004
63"
M
587
71"
N
78"
O
Figure 3. Chemotactic responses of a Dictyostelium discoideum cell to a micropipette filled with cyclic AMP (cAMP). The cell is moving towards the micropipette at the top of the image in K. In L, the pipette has been moved to the lower left and the cell reorients by retracting its protrusion at the top of the field and extending a new one towards the pipette. Time (min) shown in upper right of images. Scale bar, 20 mm. Reprinted, with permission, from Ref. [13]. q (1986) Company of Biologists.
synchronized with spontaneous oscillations in cyclic AMP (cAMP) levels. Several years earlier, cAMP had been identified as being a chemoattractant for D. discoideum by Theo Konijn and John Bonner [6]. Gerisch showed that a pulse of secreted cAMP not only induced chemotaxis in a neighboring cell but also induced this cell to secrete its own burst after a short time period, which then caused chemoattraction and cAMP production in another nearby cell. Thus, the chemoattractant signal was identical to the signal relayed in the field of aggregating cells. This concept also explained how chemotaxis could assemble cells over distances (up to 0.5 cm) that were far too large to enable sensing of a simple cAMP gradient and, thus, provided a mechanism that coupled oscillations to chemotaxis for efficient aggregation. This experimental system, in which the periodic oscillations essential for aggregation could be reproducibly generated in dense cell suspensions, enabled Gerisch to explore biochemical signaling pathways downstream of cAMP stimulation, including those involving intracellular pH changes, Ca2C fluxes, cAMP synthesis, cyclic GMP (cGMP) production and cytoskeletal regulation. With Dieter Malchow and two students in his laboratory (Werner Roos and Ursula Wick), a series of ingenious experiments overcame the difficulties posed by high phosphodiesterase activity and demonstrated both the binding of cAMP to the cell surface and the cAMPstimulated synthesis and secretion of new cAMP (a process known as cAMP relay) [7]. The role of the actomyosin cytoskeleton in cellular motility The dramatic oscillations in light scattering in response to cAMP reflect cytoskeletal rearrangements that are caused by the chemotactic response that enables D. discoideum cells to aggregate. Beginning in the 1980s and continuing to this day, Gerisch has studied the cytoskeletal changes that occur not only in the chemotactic motility of ameboid cells but also in phagocytosis, pinocytosis and mitosis, using D. discoideum as a model and combining its emerging genetics with high-resolution microscopy. An important microscopic assay for these studies grew out of an attempt with Dieter Huelser to record membranepotential changes in response to stimulation with chemoattractant. Although the membrane properties of the cells made the original goal of making membrane-potential measurements impossible, a valuable result was the www.sciencedirect.com
discovery that a micropipette filled with cAMP could be used to stimulate cells locally, enabling the direct recording of shape changes during the chemotaxis of single amebae (Figure 3). One of the early surprises that came from the genetic studies by Angelika Noegel and Michael Scheicher [8] was the apparent redundancy of components. Genetic ablation of what were thought to be major cytoskeletal proteins, such as a actinin, had only minor effects on cell movement and chemotaxis. This was surprising because it was expected that mutants lacking these proteins would show more-dramatic phenotypes. Today, the concept of redundancy is pervasive, with knockout mouse studies often finding minimal phenotypes. But the meaning of redundancy is still debated, with many researchers suggesting that, under critical conditions, no two proteins will be totally redundant. Recently, Gerisch has returned to one of his early passions – meticulous imaging studies – embarking with Annette Muller-Taubenberger on a detailed examination of the dynamics and interactions between cell-substratum adhesion and proteins such as the Arp2/3 complex and talin [9]. In addition to the elegant light-microscopic analysis of green fluorescent protein (GFP) fusion proteins, Gerisch and Igor Weber have joined forces with Wolfgang Baumeister and Ohad Medalia to show, by way of the actin cytoskeleton example, that cryoelectron tomography can be used to study the 3D topology of cellular macromolecules in situ at a resolution of 5–6 nm [10] (Figure 4). In combination with the many D. discoideum mutants lacking specific cytoskeletal proteins, this technique promises insights of unparalleled depth and detail into the cellular functions of the cytoskeleton. Still working at the cutting edge with his characteristic commitment to precision and rigor, Gerisch continues to advance our understanding of a range of biological processes, including cell motility, endocytosis, cell division and adhesion. Concluding remarks The legacy of influential scientists extends beyond the work they, themselves, have done to the impact that they have had on the careers of others in their field and the research carried out by those people – their scientific offspring, as it were. During nearly half a century of sustained effort, Guenther Gerisch has inspired colleagues with the development of new methods that enable
588
Review
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Gerisch’s own laboratory has been enormous. Gerisch’s decision to study the role of the actomyosin cytoskeleton in chemotactic motility has, thus, resulted in D. discoideum becoming the model of choice and easily the best-understood system in this area. The lessons learned from D. discoideum have contributed to the understanding of such phenomena as leukocyte chemotaxis towards wound and inflammation sites, tumor cell motility and neuronal targeting in the central nervous system. Acknowledgements We thank the many colleagues of Guenther Gerisch who contributed helpful comments and we apologize for not contacting more of Guenther’s colleagues so that they could also have contributed.
References
Figure 4. Visualization by cryoelectron tomography of the Dictyostelium discoideum actin network, membranes and cytoplasmic macromolecular complexes. A volume of 815 ! 970 ! 97 nm3 was subjected to surface rendering. Colors were subjectively attributed to linear elements to mark the actin filaments (red), other macromolecular complexes (mostly ribosomes) (green) and membranes (blue). Reprinted, with permission, from Ref. [10]. q (2002) American Association for the Advancement of Science (http://www.sciencemag.org/).
imaginative and careful experiments essential for identifying the specific contributions of individual proteins to cellular processes, combined with a broad interest in how cells work as integrated systems. Many of the D. discoideum research groups around the world, from places as far flung as Australia to the USA and various European countries, are led by people who passed through the Gerisch Cell Biology Department in Martinsried, either as PhD students or as postdoctoral researchers. Particularly in the area of nonmuscle cell motility and chemotaxis, the collective impact of their work and the ongoing work in
1 Gerisch, G. (1959) Ein submerskulturver fahren fur entwicklungsphysiologische untersuchungen an Dictyostelium discoideum. Naturwissenschaften 46, 654–656 2 Beug, H. and Gerisch, G. (1972) A micromethod for routine measurement of cell agglutination and dissociation. J. Immunol. Methods 2, 49–57 3 Beug, H. et al. (1971) Cell dissociation: univalent antibodies as a possible alternative to proteolytic enzymes. Science 173, 742–743 4 Muller, K. and Gerisch, G. (1978) A specific glycoprotein as the target site of adhesion blocking Fab in aggregating Dictyostelium cells. Nature 274, 445–449 5 Gerisch, G. and Hess, B. (1974) Cyclic-AMP-controlled oscillations in suspended Dictyostelium cells: their relation to morphogenetic cell interactions. Proc. Natl. Acad. Sci. U. S. A. 71, 2118–2122 6 Konijn, T. et al. (1968) Cyclic AMP: a naturally occurring acrasin in the cellular slime molds. Am. Nat. 102, 225–233 7 Gerisch, G. et al. (1979) Oscillations of cyclic nucleotide concentrations in relation to the excitability of Dictyostelium cells. J. Exp. Biol. 81, 33–47 8 Gerisch, G. et al. (1991) Genetic alteration of proteins in actin-based motility systems. Annu. Rev. Physiol. 53, 607–628 9 Gerisch, G. and Muller-Taubenberger, A. (2003) GFP-fusion proteins as fluorescent reporters to study organelle and cytoskeleton dynamics in chemotaxis and phagocytosis. Methods Enzymol. 361, 320–337 10 Medalia, O. et al. (2002) Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science 298, 1209–1213 11 Bretschneider, T. et al. (2004) Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr. Biol. 14, 1–10 12 Insall, R. et al. (2001) Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis. Cell Motil. Cytoskeleton 50, 115–128 13 Claviez, M. et al. (1986) Cytoskeletons from a mutant of Dictyostelium discoideum with flattened cells. J. Cell Sci. 86, 69–82
Trends in Cell Biology: new impact factor! A big thank you from the editorial team to all our readers. Thanks to you, the 2003 ISI impact factor for Trends in Cell Biology is 19.6. Trends in Cell Biology continues to be one of the most high-profile journals in which to publish! Cristina Pelizon Editor www.sciencedirect.com
Peter Chapman Assistant Editor
Helen Gannon Editorial Coordinator