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How do eucaryotic cells construct their cytoarchitecture?
Cell. Vol. 24, 4-5,
April 1981,
Copyright
0 1981
by MIT
How Do Eucaryotic Cells Construct Their Cytoarchitecture? Alice 6. Fulton Massachusetts Institute of Technology Cambridge, Massachusetts 02139
In eucaryotic cells, exquisite spatial precision is underlain by a cytoarchitecture of great complexity. Space-filling meshworks that include microtubules, microfilaments, intermediate filaments and microtrabeculae interconnect with each other and with a multitude of cellular structures such as microvilli, centrioles, ruffles and adhesion plaques. Such spatial precision and complexity raise many questions: How do cells construct their cytoarchitecture? Is there a common mechanism or central means of control over spatial order? How can such questions be asked experimentally? It appears that a mixed strategy of protein chemistry and experimental cell biology is beginning to address these questions. One classical approach to these questions is through protein chemistry. The recent successes in assembling in vitro the most stable of the cytoskeletal filaments, namely the intermediate filaments, have permitted structural analyses that highlight both the strengths and the limits of protein chemistry. Steiner et al. (PNAS 77,4534-4538, 1980) deduced a common model for the structural subunits of the intermediate filaments of baby hamster kidney cells (decamin) and of epidermal keratinocytes (keratin); this structural subunit model contains three molecules in a coiled coil, in which two long m-helical domains are joined and abutted by globular regions. Variation between different filaments is restricted to these globular domains, which vary between 5 and 17 kd. This trimeric subunit, 48 nm long and punctuated every 18 nm by a globular domain, is itself packed (somehow) in the intermediate filament proper. This subunit structure evokes functional questions and suggests experimental approaches to them: Are globular domains favored sites for interacting with other proteins? Can subunits of different IF proteins pack in one intermediate filament? This analysis of the IF subunit will be fruitful in many ways, but the analysis itself compels us to recognize that it offers no insight into the biological assembly of the subunit. The protein chemistry begins with protofilaments in 5 mM NaCI; even a slight increase in salt leads to filaments of full size. We may doubt that intracellular salt concentrations are ever low enough to foster disassembly or permit assembly from solution. The assembly and spatial control of two other major filamentous systems has been explored theoretically by Kirschner (JCB 86, 330-334, 1980). He begins with Wegner’s treadmilling theory, which proposes a steady state in which on average monomers enter filaments at one end and leave at the other, consuming ATP or GTP at some step. Such treadmilling has been observed in vitro for purified actin and tubulin; from
the observation that in cells, structural filaments rarely have two free ends, Kirschner predicts that the free end of a filament should be the fast-assembling end, to suppress indiscriminant polymerization. AS he points out, this prediction is fulfilled by most microtubular structures in the cell, but actin-containing structures (with one exception) are in the “wrong” polarity. Thus for three of four major filament systems, available theory from protein chemistry offers insight into the assembly in vivo of microtubules, is contradicted by the patterns of microfilaments and cannot address the assembly of intermediate filaments. However, there is an impressive body of experimental results rich in possible implications for eucaryotic morphogenesis. King has reviewed the control of bacteriophage assembly (Biol. Reg. Dev. 2, 101-l 32, 1980) in much deeper detail than “spontaneous assembly from solution”; many of its characteristics would be functionally relevant if true for eucaryotes. Phage assembly is strictly sequential; proteins add in sequence to a substrate that is a complex, often heterogeneous subassembly, and their association with that structure is often irreversible under physiological conditions, although the structure as a whole may undergo subtle and significant rearrangement. The stability of the structure is intrinsic, although sometimes this is the consequence of modifications, such as proteolysis; there are also proteins that are structural catalysts, participating in a structure transiently and recycling to modify other structures. In some cases, specific proteins account for geometric features such as diameters and vertices; in other cases, relative rates of synthesis may be responsible. It is striking that exquisite spatial order in phages is apparently the result of these properties; temporal controls of morphogenesis have not been seen. This rich and thought-provoking model of morphogenesis carries a sobering practical consideration. It is the sum of many years of research on experimental organisms, with known genomes, fixed spatial patterns, stoichiometric compositions and short generations, that intrinsically carry a rapid, quantitative test for fidelity of assembly. Would it be practical to apply this method of analysis to eucaryotic cells? We do not need to make the depressing calculation. It turns out that the phage studies owe their success, in part, to the fact that most phage components are globular proteins. DeRosier et al., in explaining the packing of actin filaments in the inner ear (Nature 287, 291-296, 1980). discovered a structural principle about the packing of helices, a principle that is compelling in its power and elegance and profound in its implications for the structures of eucaryotic cells.
Cell 5
Globular proteins constrained by an explicit bonding rule can form only one structure or, rarely, a few specific and predictable structures; this spatial principle led to the early success in analyzing polyhedral viruses and underlies the successes with phage morphogenesis. In great and surprising contrast, helical structures have no such geometric predictability. A bonding rule between helices as explicit and constraining as any in an icosahedral virus can produce packing in any pattern-triangular, square, hexagonal or even liquid; this freedom in packing results because the helix contains a multitude of equivalent sites. Thus bonds made at equivalent sites on a helix will pack the helices differently, depending on how far up the helix the next bond is made. This result is general for all helices and can be directly extended to packing in three directions (they consider the case of two) by including bonds that produce skewed, instead of parallel, associations. The power and generality of this finding can hardly be overemphasized. Microfilaments, microtublules, intermediate filaments, collagen, cellulose and chromatin itself are among the biologically significant helices that show spatial order in eucaryotic cells; this argument shows that knowledge of local behavior, such as rules of bonding, cannot predict the overall structure or pattern. In protein chemistry, the indeterminacy of helical packing places an absolute limit to knowledge, a limit in principle. Protein chemistry can tell us, and has already told us, many fundamental properties of structural components; but it is, in principle, unable to tell the whole story. How, then, does spatial specificity arise? And how can it be explored experimentally? DeRosier and his coworkers (op. cit.) suggest briefly that it is “the conditions at the time of assembly” that determine pattern; this is related in a deep sense to the fact that only cells make cells. It seems likely that the intrinsic limitations of protein chemistry will only be overcome by including the behavior of the cell itself in the analysis. Several lines of experiments have begun to follow this mixed strategy involving reciprocation between protein chemistry and function in a living cell. Microtubules assembling in a living cell have been followed by fluorescence microscopy after living cells were microinjected with fluorescent tubulin 4Keith et al., JCB 88, 234-240, 1981). Fluorescence is incorporated first at the periphery (as Kirschner’s model suggested for microtubules) and with time becomes localized in fibers throughout much of the cell. Assem-
bly is specific; denatured tubulin ends in autophagic vacuoles and colcemid delocalized the fluorescence. There is an interesting difference in kinetics; in vitro, assembly is complete in about 5 min, but it requires about 2 hr in the cell. The significance of these kinetic differences will be clearer when quantitation is possible. Also possibly significant is the lack of incorporation of exogenous microtubule-associated proteins (MAPS); these MAPS are competent in vitro, so that some aspect of the injected cell must account for lack of incorporation. These particular MAPS are derived from brain; microinjection into neuroblastoma cells might be informative. These experiments show, in any case, that with enough care even a fragile protein like tubulin can be purified and chemically modified to probe the assembly of structures in the living cell. The many demonstrations that cytoskeletons can be freed of soluble proteins by gentle extraction made possible a different examination of assembly in vivo (Fulton et al., Cell 20, 849-857, 1980). Combining a gentle extraction procedure and autoradiography after a brief pulse of methionine made it clear that, in 3T3 cells, many cytoskeletal proteins associate with the skeleton close to the time and place of their synthesis. Given time, the proteins rearrange in the skeleton, but only during continuing protein synthesis. The extraction removed m’icrotubules, so that in many ways the skeletons examined here are complements of the structures shown by tubulin fluorescence; thus it is not surprising that 3 hr saw little incorporation into the skeleton from the soluble phase. Finally, a satisfactory explanation of how cells construct their cytoarchitecture must account for cell behavior such as that seen by Solomon (Cell 2 1,333338, 1980). Sister neuroblastoma cells often share a particular morphology; this shape can be erased with Nocodazole, which depolymerizes microtubules. When the drug is washed out, cells reexpress a detailed morphology. This morphology is often the morphology seen before drug treatment. Even cells that move during reexpression still accurately regenerate their shapes. Clearly, a complete account of cell selfconstruction must include such detailed, persistent and global patterns in its explanation. Protein chemistry by itself, though rich in insights, would not in practice and cannot in principle supply a complete description of the remarkably specific patterns of the eucaryotic cell; however, when closely joined to experimental approaches to cell behavior, it is beginning to yield understanding of this multiplex and central biological structure.
April 1981,
Copyright
0 1981
by MIT
How Do Eucaryotic Cells Construct Their Cytoarchitecture? Alice 6. Fulton Massachusetts Institute of Technology Cambridge, Massachusetts 02139
In eucaryotic cells, exquisite spatial precision is underlain by a cytoarchitecture of great complexity. Space-filling meshworks that include microtubules, microfilaments, intermediate filaments and microtrabeculae interconnect with each other and with a multitude of cellular structures such as microvilli, centrioles, ruffles and adhesion plaques. Such spatial precision and complexity raise many questions: How do cells construct their cytoarchitecture? Is there a common mechanism or central means of control over spatial order? How can such questions be asked experimentally? It appears that a mixed strategy of protein chemistry and experimental cell biology is beginning to address these questions. One classical approach to these questions is through protein chemistry. The recent successes in assembling in vitro the most stable of the cytoskeletal filaments, namely the intermediate filaments, have permitted structural analyses that highlight both the strengths and the limits of protein chemistry. Steiner et al. (PNAS 77,4534-4538, 1980) deduced a common model for the structural subunits of the intermediate filaments of baby hamster kidney cells (decamin) and of epidermal keratinocytes (keratin); this structural subunit model contains three molecules in a coiled coil, in which two long m-helical domains are joined and abutted by globular regions. Variation between different filaments is restricted to these globular domains, which vary between 5 and 17 kd. This trimeric subunit, 48 nm long and punctuated every 18 nm by a globular domain, is itself packed (somehow) in the intermediate filament proper. This subunit structure evokes functional questions and suggests experimental approaches to them: Are globular domains favored sites for interacting with other proteins? Can subunits of different IF proteins pack in one intermediate filament? This analysis of the IF subunit will be fruitful in many ways, but the analysis itself compels us to recognize that it offers no insight into the biological assembly of the subunit. The protein chemistry begins with protofilaments in 5 mM NaCI; even a slight increase in salt leads to filaments of full size. We may doubt that intracellular salt concentrations are ever low enough to foster disassembly or permit assembly from solution. The assembly and spatial control of two other major filamentous systems has been explored theoretically by Kirschner (JCB 86, 330-334, 1980). He begins with Wegner’s treadmilling theory, which proposes a steady state in which on average monomers enter filaments at one end and leave at the other, consuming ATP or GTP at some step. Such treadmilling has been observed in vitro for purified actin and tubulin; from
the observation that in cells, structural filaments rarely have two free ends, Kirschner predicts that the free end of a filament should be the fast-assembling end, to suppress indiscriminant polymerization. AS he points out, this prediction is fulfilled by most microtubular structures in the cell, but actin-containing structures (with one exception) are in the “wrong” polarity. Thus for three of four major filament systems, available theory from protein chemistry offers insight into the assembly in vivo of microtubules, is contradicted by the patterns of microfilaments and cannot address the assembly of intermediate filaments. However, there is an impressive body of experimental results rich in possible implications for eucaryotic morphogenesis. King has reviewed the control of bacteriophage assembly (Biol. Reg. Dev. 2, 101-l 32, 1980) in much deeper detail than “spontaneous assembly from solution”; many of its characteristics would be functionally relevant if true for eucaryotes. Phage assembly is strictly sequential; proteins add in sequence to a substrate that is a complex, often heterogeneous subassembly, and their association with that structure is often irreversible under physiological conditions, although the structure as a whole may undergo subtle and significant rearrangement. The stability of the structure is intrinsic, although sometimes this is the consequence of modifications, such as proteolysis; there are also proteins that are structural catalysts, participating in a structure transiently and recycling to modify other structures. In some cases, specific proteins account for geometric features such as diameters and vertices; in other cases, relative rates of synthesis may be responsible. It is striking that exquisite spatial order in phages is apparently the result of these properties; temporal controls of morphogenesis have not been seen. This rich and thought-provoking model of morphogenesis carries a sobering practical consideration. It is the sum of many years of research on experimental organisms, with known genomes, fixed spatial patterns, stoichiometric compositions and short generations, that intrinsically carry a rapid, quantitative test for fidelity of assembly. Would it be practical to apply this method of analysis to eucaryotic cells? We do not need to make the depressing calculation. It turns out that the phage studies owe their success, in part, to the fact that most phage components are globular proteins. DeRosier et al., in explaining the packing of actin filaments in the inner ear (Nature 287, 291-296, 1980). discovered a structural principle about the packing of helices, a principle that is compelling in its power and elegance and profound in its implications for the structures of eucaryotic cells.
Cell 5
Globular proteins constrained by an explicit bonding rule can form only one structure or, rarely, a few specific and predictable structures; this spatial principle led to the early success in analyzing polyhedral viruses and underlies the successes with phage morphogenesis. In great and surprising contrast, helical structures have no such geometric predictability. A bonding rule between helices as explicit and constraining as any in an icosahedral virus can produce packing in any pattern-triangular, square, hexagonal or even liquid; this freedom in packing results because the helix contains a multitude of equivalent sites. Thus bonds made at equivalent sites on a helix will pack the helices differently, depending on how far up the helix the next bond is made. This result is general for all helices and can be directly extended to packing in three directions (they consider the case of two) by including bonds that produce skewed, instead of parallel, associations. The power and generality of this finding can hardly be overemphasized. Microfilaments, microtublules, intermediate filaments, collagen, cellulose and chromatin itself are among the biologically significant helices that show spatial order in eucaryotic cells; this argument shows that knowledge of local behavior, such as rules of bonding, cannot predict the overall structure or pattern. In protein chemistry, the indeterminacy of helical packing places an absolute limit to knowledge, a limit in principle. Protein chemistry can tell us, and has already told us, many fundamental properties of structural components; but it is, in principle, unable to tell the whole story. How, then, does spatial specificity arise? And how can it be explored experimentally? DeRosier and his coworkers (op. cit.) suggest briefly that it is “the conditions at the time of assembly” that determine pattern; this is related in a deep sense to the fact that only cells make cells. It seems likely that the intrinsic limitations of protein chemistry will only be overcome by including the behavior of the cell itself in the analysis. Several lines of experiments have begun to follow this mixed strategy involving reciprocation between protein chemistry and function in a living cell. Microtubules assembling in a living cell have been followed by fluorescence microscopy after living cells were microinjected with fluorescent tubulin 4Keith et al., JCB 88, 234-240, 1981). Fluorescence is incorporated first at the periphery (as Kirschner’s model suggested for microtubules) and with time becomes localized in fibers throughout much of the cell. Assem-
bly is specific; denatured tubulin ends in autophagic vacuoles and colcemid delocalized the fluorescence. There is an interesting difference in kinetics; in vitro, assembly is complete in about 5 min, but it requires about 2 hr in the cell. The significance of these kinetic differences will be clearer when quantitation is possible. Also possibly significant is the lack of incorporation of exogenous microtubule-associated proteins (MAPS); these MAPS are competent in vitro, so that some aspect of the injected cell must account for lack of incorporation. These particular MAPS are derived from brain; microinjection into neuroblastoma cells might be informative. These experiments show, in any case, that with enough care even a fragile protein like tubulin can be purified and chemically modified to probe the assembly of structures in the living cell. The many demonstrations that cytoskeletons can be freed of soluble proteins by gentle extraction made possible a different examination of assembly in vivo (Fulton et al., Cell 20, 849-857, 1980). Combining a gentle extraction procedure and autoradiography after a brief pulse of methionine made it clear that, in 3T3 cells, many cytoskeletal proteins associate with the skeleton close to the time and place of their synthesis. Given time, the proteins rearrange in the skeleton, but only during continuing protein synthesis. The extraction removed m’icrotubules, so that in many ways the skeletons examined here are complements of the structures shown by tubulin fluorescence; thus it is not surprising that 3 hr saw little incorporation into the skeleton from the soluble phase. Finally, a satisfactory explanation of how cells construct their cytoarchitecture must account for cell behavior such as that seen by Solomon (Cell 2 1,333338, 1980). Sister neuroblastoma cells often share a particular morphology; this shape can be erased with Nocodazole, which depolymerizes microtubules. When the drug is washed out, cells reexpress a detailed morphology. This morphology is often the morphology seen before drug treatment. Even cells that move during reexpression still accurately regenerate their shapes. Clearly, a complete account of cell selfconstruction must include such detailed, persistent and global patterns in its explanation. Protein chemistry by itself, though rich in insights, would not in practice and cannot in principle supply a complete description of the remarkably specific patterns of the eucaryotic cell; however, when closely joined to experimental approaches to cell behavior, it is beginning to yield understanding of this multiplex and central biological structure.