Journal of Neurocytology 7, 313-321 (1978)
A Mg2+-dependent class of thick filaments and correlated nuclear chromatin condensation in catfish photoreceptors THOMAS
RYAN
and H .
DAVID
POTTER
Center f o r Neural Sciences, Indiana University, Bloomington, Indiana 4 7401, U.S.A.
Received 30 September 1977; revised 22 November 1977; accepted 28 November 1977
Summary Photoreceptor cells of excised catfish retinae show morphological differences when incubated in Ringer's solutions of varying ionic composition. Two striking changes were observed in photoreceptor cells incubated in a high Mg 2+ (25 mM) Ringer's: (1) Thick filaments appeared in the cytoplasm of receptor terminals and myoids; ( 2 ) A pronounced condensation of nuclear chromatin occurred in certain nuclei in the outer nuclear layer. The filaments occurred in lattices or bundles. The bundles had a diameter of approximately 0 . 0 5 - 0 . 2 / a m and had either tapered or frayed ends. They were observed with somewhat higher incidence in tissue incubated in a 25 ~ M Mg2+ Ringer's with EGTA added to chelate Ca 2+. A common basis for the cytoplasmic and nuclear changes may lie in a redistribution of fibrous proteins brought about by the increased Mg 2+ concentration.
Introduction
In the course of experimental studies on the organization and relative stability of cytoplasmic microtubule and filament systems in nerve cells, we have found an unexpected class of complex filaments in photoreceptor terminals and myoids. In this report, we will describe: (a)the distribution and morphology of these novel filaments which appear following in vitro incubation in media containing elevated Mg2+ levels; and (b) the unusual condensation of nuclear chromatin produced under the same conditions in part of the photoreceptor cells. Materials and methods Light- and dark-adapted channel catfish (Ictalurus punctatus) were lightly anaesthetized in 0.1% tricaine methanesulphonate (Ayerst Laboratories, Inc., New York). The eyes were removed and the vitreous b o d y flushed from the posterior chamber. The tissue was incubated in one of the following oxygenated Ringer's solutions for 1 h at 21 ~ C: Condition A (standard Ringer's): 9 1978 Chapman and Hall Ltd. Printed in Great Britain
314
RYAN and POTTER
100mM NaC1, 2.5mM KC1, 1.5mM CaCl2, 5mM NaHCO3, 0.5 mM NaH2PO4, 1ram MgCI2; Condition B: Same as A, except the MgC12 concentration was increased to 25 mM; Condition C: Same as B, except no CaC12, and with 5 mM EGTA [ethylene glycol-bis(B-aminoethyl ether) N,N'-tetraacetic acid, (Calbiochem, La Jolla, California); Condition D: Same as A, except no CaC12, no MgC12, and with 1 mM EDTA [(ethylene-dinitrilo)-tetraacetic acid, disodium salt, (Matheson, Coleman, and Bell, Norwood, Ohio)]. The tissue was fixed in 1% formaldehyde (prepared from paraformaldehyde) and 1% glutaraldehyde (charcoal filtered, Fisher Scientific Co., Fair Lawn, New Jersey) in 0.1 M cacodylate buffer at pH 7.3 (Karnovsky, 1965), immersed in buffered 1% osmium tetroxide (Stevens Metallurgical Corp., New York) for 1.75 h and then stained en bloc in 2% uranyl acetate (Mallinckrodt Chemical Works, St. Louis, Missouri) in 0.05 M maleate buffer at pH 5.2 at 4~ for 1.75 h (Karnovsky, 1967). The tissue was dehydrated in graded acetones and embedded in Araldite 502 (Ernest F. Fullam, Inc., Schenectady, New York). Sections were cut on a Sorvall Porter-Blum MT-2 ultramicrotome, stained with uranyl acetate and lead citrate (Ladd Research Industries, Inc., Burlington, Vermont), carbon-coated and examined on a Philips 201 electron microscope.
Observations
For each incubation condition, the morphology of the retina was examined with specific regard to two features: (a) presence of filaments in photoreceptor cell terminals and myoids; and (b) chromatin condensation in cell nuclei. In every case, the general morphology of the tissue and the quality of fixation were evaluated. On the basis of these observations, qualitative differences were noted. We have correlated these morphological differences with the variations in divalent ion concentration. Thick Filaments Close examination of retinal receptor terminals incubated under certain conditions revealed thick filaments situated among the synaptic vesicles (Figs. 1-5). Similar structures were also detected in receptor myoids (Figs. 6--10). They were observed following incubation in a high Mg2+ Ringer's solution (Condition B) and with slightly higher incidence in a similar solution with EGTA (Condition C). Their distribution was somewhat uneven. In some sections thick filaments appeared in nearly every photoreceptor terminal, but in other sections they were rare.
Figs. 1 - 5 are electron micrographs of retinal photoreceptor terminals cut in longitudinal section following incubation in high Mg2+, EGTA Ringer's solution (Condition C). Fig. 1. Thick filaments situated among the synaptic vesicles illustrating parallel or branching orientation and complex organization, x 37 000. Fig. 2. High magnification of a thick filamentous bundle revealing internal structure and frayed ends. x 92 000. Fig. 3. Thick filaments lying close to and parallel with the plasma membrane (arrows). x 96 000. Fig. 4. Thick filaments interlacing with one another, x 69 000. Fig. 5. A thick filamentous bundle running parallel to the plasma membrane (arrows), displays tapering at both ends. x 69 000.
316
RYAN and POTTER
The filamentous structures consist of bundles, 0 . 0 5 - 0 . 2 t~m in diameter, of parallel filaments that either taper or fray at the ends. The length of the bundles varied considerably. In the terminal, the thick filaments seemed to assume a random orientation, both relative to the terminal membrane and to each other. This randomness may account, at least in part, for the observed variability in filament length. In the myoid, the filaments were usually oriented parallel to the long axis of the photoreceptor cell. The filaments were found in terminals containing multiple (cone) or single (rod) synaptic ribbons. They were present in both light- and dark-adapted retinae. Individual thick filaments often had a dense segment 2 0 - 3 0 nm in diameter by 0 . 1 - 0 . 2 tim in length that gave way at each end to less dense continuations. These then either tapered to points or broadened to 3 0 - 4 0 nm and interlaced with neighbouring filaments. The thick filaments sometimes lay adjacent to microfilament bundles (Figs. 6 and 8) or microtubules (Figs. 8 and 10). The elevated Mg 2+ conditions had no apparent effects on microfilaments or microtubules in the myoid, ellipsoid, or calyceal processes.
Chromatin Condensation The outer nuclear layer contains the nuclei of receptor cells organized into two parallel layers. The layer closer to the outer segments is composed of cone nuclei. The layer closer to the outer plexiform layer is composed of rod nuclei. In tissue fixed without incubation, the two kinds of nuclei are virtually indistinguishable on the basis of chromatin organization (Fig. 11). Their chromatin appears evenly dispersed throughout the nucleus. In the experimental conditions, condensation was observed, to varying degrees, when the incubation media contained a relatively high concentration of Mg 2+ (25 raM; Fig. 12). This phenomenon occurred in nearly all nuclei in the outer (cone) layer, but was rare in the inner (rod) layer. It did not occur in the nuclei of any other cell type in the retina. Condensation did not occur when Mg 2+ was present in physiological concentrations (Condition A) nor when it was removed by EDTA (Condition D). The presence or absence of Ca z+ seemed to have no effect on chromatin organization. Figs. 6--10 are electron micrographs of photoreceptor myoids sectioned longitudinally after incubation in high Mg2+, EGTA Ringer's solution (Condition C). mf, mierofilament; mt, microtubule. Fig. 6. Long, thick filamentous arrays parallel to the plasma membrane (arrows). Note the adjacent microfilament bundle, x 40 000. Fig. 7. A thick filament bundle near the nucleus, x 57 000. Fig. 8. Complex filament arrays adjacent to microtubules and bundles of microfilaments. x 60 000. Fig. 9. A grazing section through a thick filament bundle at high magnification shows the characteristic sparse packing, x 98 000. Fig. 10. A thick filament bundle showing frayed ends and sparse packing in close proximity to a microtubule, x 60 000.
9lgZ+-dependent changes in catfish photoreceptors
319
)iscussion Ne have reported here on the discovery of a Mg2+-dependent population of thick :ilamentous structures in photoreceptor cells. Their molecular composition and :ytological significance is unknown, but their characteristics may be discussed in ight of recent findings in the biology of intracellular fibrous proteins. A type of thick filament in photoreceptor axons of saltwater teleosts was 7eported by Burnside (1976). These filaments averaged 14 nm in diameter with ;idearms at 15 nm intervals. They were present in ribbon-like clusters and were 3reserved only in certain fixation conditions. The filaments in catfish photo7eceptors reported here occurred in different parts of the photoreceptor cell and ~r Mg2+-dependent. A comparison of structural details indicated that the Viga+-induced filaments were not of uniform diameter and did not show periodic ;ide arms. Both types of filaments were sensitive to fixation or incubation :0nditions. Morphologically, the Mg2+-induced filaments bear some resemblance to myosin md myosin-like filaments in smooth muscle (Rosenbluth, 1971a) or in other non-striated muscle preparations (Schoenberg, 1969; Rosenbluth, 1971b; Stossel md Pollard, 1973; Niederman and Pollard, 1975). Schoenberg (1969) and Stossel md Pollard (1973) have demonstrated the requirement for cations for in vitro nyosin filament formation. The thick filaments described here are also sensitive to ]ivalent cation concentrations, and, in fact, require elevated Mg 2+ to aggregate into irrays that are observable with the electron microscope. Actin is a possible constituent of the thick filaments. This protein has been shown -o exist in unpolymerized form in many non-muscle cells (Bray and Thomas, 1976). Vlicrofilaments ( 5 - 7 nm in diameter) are now believed to be a polymerized, !ilamentous form of actin on the basis of arrowhead formation with heavy neromyosin in glycerinated cells. Bundles of microfilaments were usually present in :he myoid and calyceal processes of catfish photoreceptors (also see Burnside, [976), but no consistent relationship was apparent between these bundles and the :hick filaments. Any actin within the thick filaments would represent an alternate !orm aggregated in elevated Mg 2+. Actin aggregation in elevated Mg 2+ in vitro has 3een reported (Cooke et al., 1976), but the resulting paracrystals and F-actin 3undles did not appear morphologically similar to the thick filaments. It is probable :hat the fuzzy filamentous material adhering to and interspersed between synaptic lesicles (Figs. 1--5) represents an aggregated protein system that includes actin
qgs. 11 and 12 are low magnification micrographs of the outer nuclear and outer plexiform layers. :n, cone nuclei; rn, rod nuclei; pt, photoreceptor terminal. ~ig. 11. Nuclear regions following incubation in standard Ringer's solution (Condition A). The mclei in the two layers are virtually indistinguishable, x 7500. qg. 12. Nuclear regions following incubation in high Mg2+, EGTA Ringer's solution Condition C). The outer (cone) layer of nuclei have condensed chromatin, whereas in the inner rod) nuclei the chromatin is dispersed, x 7500.
320
RYAN and POTTER
(LeBeux and Willemot, 1975; Puszkin et al., 1976). The thick filaments appeared in intimate association with this material and seemed to be morphologically continuous with it, as were the synaptic densities and ribbons. The role of thick filaments or their antecendents in the photoreceptor cell is as yet unknown. It seems noteworthy, however, that their presence appeared to be confined to the terminal and myoid. Both these areas perform roles that could conceivably involve the interaction of filamentous arrays for dynamic structural operations. Thus the myoid contracts to change the relative position of the outer segment in response to fluctuating levels of light intensity. The size and structural complexity of the receptor cell terminal may necessitate the use of filamentous systems both for preserving its structural integrity and for participating in a cycle of exocytosis and endocytosis in transmitter release. It is probable that interactions between various populations of filamentous proteins are involved in these dynamic processes. The Mg2*-induced thick filaments described here may represent or arise from such dynamic populations. The phenomenon of chromatin condensation in the outer layer of receptor cell nuclei after incubation in high Mg2§ Ringer's solutions may also be related to the state of aggregation of filamentous proteins within the nucleus. Lestourgeon et al. (1975) have isolated two non-histone proteins from the Myxomycete Physarum and identified them as actin and myosin. They suggest that these contractile proteins may be involved in the process of heterochromatization. They state that the role of Mg 2§ in this event is unclear, although it may be required for actomyosin binding proteins to inhibit the Ca2§ ATPase activity of the actomyosin. Other investigators have suggested that ATP may cause the decondensation of chromatin by acting as a Mg2§ chelating agent (Leake et al., 1972; Comings and Okada, 1976). These results, together with those reported here, suggest a role for filamentous proteins in the condensation of nuclear chromatin. It can be further proposed that this interaction is somehow mediated by Mg2§ ions. We are, however, unable to offer an explanation for the apparent confinement of this phenomenon to the outer layer of nuclei. Because the two layers are believed to segregate into rod and cone nuclei and because it is the cones (outer layer) that are most active in the light-adapted state, we attempted to reverse the condensation by using dark-adapted retinae. No correlation with the ~tate of adaptation was found, since under these conditions, condensed chromatin was still confined to the outer layer of nuclei.
Acknowledgements This investigation was supported by National Institutes of Health grant NS 08964. The authors gratefully acknowledge Dr Gary Hafner's careful review of the manuscript and the expert technical assistance provided by Ms Betsy Osborne and Ms Kitty Johansen.
Mg2+-dependent changes in catfish photoreceptors
321
References BRAY, D. and THOMAS, C. (1976) Unpolymerized actin in tissue cells. In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 3 (edited by GOLDMAN, R., POLLARD, T., and ROSENBAUM, J.), pp. 461--73. BURNSIDE, B. (1976) Microtubules and actin filaments in teleost visual cone elongation and contraction. Journal of Supramolecular Structure 5 , 2 5 7 - 7 5 . COMINGS, D.E. and OKADA, T.A. (1976) Fine structure of the heterochromatin of the kangaroo rat Dipidomys ordii, and examination of the possible role of actin and myosin in heterochromatin condensation. Journal of Cell Science 2 1 , 4 6 5 - 7 7 . COOKE, R., CLARKE, M., yon WEDEL, R. J. and SPUDICH, J. A. (1976) Supramolecular forms of Dictyostelium actin. In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 3 (edited by GOLDMAN, R., POLLARD, T., and ROSENBAUM, J.), pp. 5 7 5 - 8 7 . KARNOVSKY, M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell Biology 27, 1 3 7 A - 1 3 8 A . KARNOVSKY, M.J. (1967) The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. Journal of Cell Biology 3 5 , 2 1 3 - 3 6 . LEAKE, R. E., TRENCH, M. E. and BARRY, J. M. (1972) Effect of cations on the condensation of hen erythrocyte nuclei and its relation to gene activation. Experimental Cell Research 71, 17-26. LEBEUX, Y. J. and WILLEMOT, J. (1975) An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. II. The synapses. Cell and Tissue Research 160, 37--68. LESTOURGEON, W.M., FORER, A., YANG, Y., BERTRAM, J. S. and RUSCH, H. P. (1975) Contractile proteins: Major components of nuclear and chromosome non-histone proteins. Biochimica et Biophysica Acta, 379, 5 2 9 - 5 2 . NIEDERMAN, R. and POLLARD, T. D. (1975) Human platelet myosin. II. In vitro assembly and structure of myosin filaments. Journal of Cell Biology 67, 7 9 - 9 2 . PUSZKIN, S., KOCHWA, S. and ROSENFIELD, R.E. (1976) Ultrastructural evidence of association of contractile proteins in synaptosomes. In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 3 (edited by GOLDMAN, R., POLLARD, T. and ROSENBAUM, J.), pp. 6 6 5 - 7 0 . ROSENBLUTH, J. (1971a) Myosin-like aggregates in trypsin-treated smooth muscle cells. Journal of Cell Biology 48, 1 7 4 - 8 8 . ROSENBLUTH, J. (1971b) Myosin-like tactoids in trypsin-treated blood platelets. Journal of Cell Biology 50, 9 0 0 - 4 . SCHOENBERG, C. F. (1969) An electron microscope study of the influence of divalent ions on myosin filament formation in chicken gizzard extracts and homogenates. Tissue and Cell 1, 83-96. STOSSEL, T. P. and POLLARD, T. D. (1973) Myosin in polymorphonuclear leukocytes. Journal of Biological Chemistry 248, 8 2 8 8 - 9 4 .
A Mg2+-dependent class of thick filaments and correlated nuclear chromatin condensation in catfish photoreceptors THOMAS
RYAN
and H .
DAVID
POTTER
Center f o r Neural Sciences, Indiana University, Bloomington, Indiana 4 7401, U.S.A.
Received 30 September 1977; revised 22 November 1977; accepted 28 November 1977
Summary Photoreceptor cells of excised catfish retinae show morphological differences when incubated in Ringer's solutions of varying ionic composition. Two striking changes were observed in photoreceptor cells incubated in a high Mg 2+ (25 mM) Ringer's: (1) Thick filaments appeared in the cytoplasm of receptor terminals and myoids; ( 2 ) A pronounced condensation of nuclear chromatin occurred in certain nuclei in the outer nuclear layer. The filaments occurred in lattices or bundles. The bundles had a diameter of approximately 0 . 0 5 - 0 . 2 / a m and had either tapered or frayed ends. They were observed with somewhat higher incidence in tissue incubated in a 25 ~ M Mg2+ Ringer's with EGTA added to chelate Ca 2+. A common basis for the cytoplasmic and nuclear changes may lie in a redistribution of fibrous proteins brought about by the increased Mg 2+ concentration.
Introduction
In the course of experimental studies on the organization and relative stability of cytoplasmic microtubule and filament systems in nerve cells, we have found an unexpected class of complex filaments in photoreceptor terminals and myoids. In this report, we will describe: (a)the distribution and morphology of these novel filaments which appear following in vitro incubation in media containing elevated Mg2+ levels; and (b) the unusual condensation of nuclear chromatin produced under the same conditions in part of the photoreceptor cells. Materials and methods Light- and dark-adapted channel catfish (Ictalurus punctatus) were lightly anaesthetized in 0.1% tricaine methanesulphonate (Ayerst Laboratories, Inc., New York). The eyes were removed and the vitreous b o d y flushed from the posterior chamber. The tissue was incubated in one of the following oxygenated Ringer's solutions for 1 h at 21 ~ C: Condition A (standard Ringer's): 9 1978 Chapman and Hall Ltd. Printed in Great Britain
314
RYAN and POTTER
100mM NaC1, 2.5mM KC1, 1.5mM CaCl2, 5mM NaHCO3, 0.5 mM NaH2PO4, 1ram MgCI2; Condition B: Same as A, except the MgC12 concentration was increased to 25 mM; Condition C: Same as B, except no CaC12, and with 5 mM EGTA [ethylene glycol-bis(B-aminoethyl ether) N,N'-tetraacetic acid, (Calbiochem, La Jolla, California); Condition D: Same as A, except no CaC12, no MgC12, and with 1 mM EDTA [(ethylene-dinitrilo)-tetraacetic acid, disodium salt, (Matheson, Coleman, and Bell, Norwood, Ohio)]. The tissue was fixed in 1% formaldehyde (prepared from paraformaldehyde) and 1% glutaraldehyde (charcoal filtered, Fisher Scientific Co., Fair Lawn, New Jersey) in 0.1 M cacodylate buffer at pH 7.3 (Karnovsky, 1965), immersed in buffered 1% osmium tetroxide (Stevens Metallurgical Corp., New York) for 1.75 h and then stained en bloc in 2% uranyl acetate (Mallinckrodt Chemical Works, St. Louis, Missouri) in 0.05 M maleate buffer at pH 5.2 at 4~ for 1.75 h (Karnovsky, 1967). The tissue was dehydrated in graded acetones and embedded in Araldite 502 (Ernest F. Fullam, Inc., Schenectady, New York). Sections were cut on a Sorvall Porter-Blum MT-2 ultramicrotome, stained with uranyl acetate and lead citrate (Ladd Research Industries, Inc., Burlington, Vermont), carbon-coated and examined on a Philips 201 electron microscope.
Observations
For each incubation condition, the morphology of the retina was examined with specific regard to two features: (a) presence of filaments in photoreceptor cell terminals and myoids; and (b) chromatin condensation in cell nuclei. In every case, the general morphology of the tissue and the quality of fixation were evaluated. On the basis of these observations, qualitative differences were noted. We have correlated these morphological differences with the variations in divalent ion concentration. Thick Filaments Close examination of retinal receptor terminals incubated under certain conditions revealed thick filaments situated among the synaptic vesicles (Figs. 1-5). Similar structures were also detected in receptor myoids (Figs. 6--10). They were observed following incubation in a high Mg2+ Ringer's solution (Condition B) and with slightly higher incidence in a similar solution with EGTA (Condition C). Their distribution was somewhat uneven. In some sections thick filaments appeared in nearly every photoreceptor terminal, but in other sections they were rare.
Figs. 1 - 5 are electron micrographs of retinal photoreceptor terminals cut in longitudinal section following incubation in high Mg2+, EGTA Ringer's solution (Condition C). Fig. 1. Thick filaments situated among the synaptic vesicles illustrating parallel or branching orientation and complex organization, x 37 000. Fig. 2. High magnification of a thick filamentous bundle revealing internal structure and frayed ends. x 92 000. Fig. 3. Thick filaments lying close to and parallel with the plasma membrane (arrows). x 96 000. Fig. 4. Thick filaments interlacing with one another, x 69 000. Fig. 5. A thick filamentous bundle running parallel to the plasma membrane (arrows), displays tapering at both ends. x 69 000.
316
RYAN and POTTER
The filamentous structures consist of bundles, 0 . 0 5 - 0 . 2 t~m in diameter, of parallel filaments that either taper or fray at the ends. The length of the bundles varied considerably. In the terminal, the thick filaments seemed to assume a random orientation, both relative to the terminal membrane and to each other. This randomness may account, at least in part, for the observed variability in filament length. In the myoid, the filaments were usually oriented parallel to the long axis of the photoreceptor cell. The filaments were found in terminals containing multiple (cone) or single (rod) synaptic ribbons. They were present in both light- and dark-adapted retinae. Individual thick filaments often had a dense segment 2 0 - 3 0 nm in diameter by 0 . 1 - 0 . 2 tim in length that gave way at each end to less dense continuations. These then either tapered to points or broadened to 3 0 - 4 0 nm and interlaced with neighbouring filaments. The thick filaments sometimes lay adjacent to microfilament bundles (Figs. 6 and 8) or microtubules (Figs. 8 and 10). The elevated Mg 2+ conditions had no apparent effects on microfilaments or microtubules in the myoid, ellipsoid, or calyceal processes.
Chromatin Condensation The outer nuclear layer contains the nuclei of receptor cells organized into two parallel layers. The layer closer to the outer segments is composed of cone nuclei. The layer closer to the outer plexiform layer is composed of rod nuclei. In tissue fixed without incubation, the two kinds of nuclei are virtually indistinguishable on the basis of chromatin organization (Fig. 11). Their chromatin appears evenly dispersed throughout the nucleus. In the experimental conditions, condensation was observed, to varying degrees, when the incubation media contained a relatively high concentration of Mg 2+ (25 raM; Fig. 12). This phenomenon occurred in nearly all nuclei in the outer (cone) layer, but was rare in the inner (rod) layer. It did not occur in the nuclei of any other cell type in the retina. Condensation did not occur when Mg 2+ was present in physiological concentrations (Condition A) nor when it was removed by EDTA (Condition D). The presence or absence of Ca z+ seemed to have no effect on chromatin organization. Figs. 6--10 are electron micrographs of photoreceptor myoids sectioned longitudinally after incubation in high Mg2+, EGTA Ringer's solution (Condition C). mf, mierofilament; mt, microtubule. Fig. 6. Long, thick filamentous arrays parallel to the plasma membrane (arrows). Note the adjacent microfilament bundle, x 40 000. Fig. 7. A thick filament bundle near the nucleus, x 57 000. Fig. 8. Complex filament arrays adjacent to microtubules and bundles of microfilaments. x 60 000. Fig. 9. A grazing section through a thick filament bundle at high magnification shows the characteristic sparse packing, x 98 000. Fig. 10. A thick filament bundle showing frayed ends and sparse packing in close proximity to a microtubule, x 60 000.
9lgZ+-dependent changes in catfish photoreceptors
319
)iscussion Ne have reported here on the discovery of a Mg2+-dependent population of thick :ilamentous structures in photoreceptor cells. Their molecular composition and :ytological significance is unknown, but their characteristics may be discussed in ight of recent findings in the biology of intracellular fibrous proteins. A type of thick filament in photoreceptor axons of saltwater teleosts was 7eported by Burnside (1976). These filaments averaged 14 nm in diameter with ;idearms at 15 nm intervals. They were present in ribbon-like clusters and were 3reserved only in certain fixation conditions. The filaments in catfish photo7eceptors reported here occurred in different parts of the photoreceptor cell and ~r Mg2+-dependent. A comparison of structural details indicated that the Viga+-induced filaments were not of uniform diameter and did not show periodic ;ide arms. Both types of filaments were sensitive to fixation or incubation :0nditions. Morphologically, the Mg2+-induced filaments bear some resemblance to myosin md myosin-like filaments in smooth muscle (Rosenbluth, 1971a) or in other non-striated muscle preparations (Schoenberg, 1969; Rosenbluth, 1971b; Stossel md Pollard, 1973; Niederman and Pollard, 1975). Schoenberg (1969) and Stossel md Pollard (1973) have demonstrated the requirement for cations for in vitro nyosin filament formation. The thick filaments described here are also sensitive to ]ivalent cation concentrations, and, in fact, require elevated Mg 2+ to aggregate into irrays that are observable with the electron microscope. Actin is a possible constituent of the thick filaments. This protein has been shown -o exist in unpolymerized form in many non-muscle cells (Bray and Thomas, 1976). Vlicrofilaments ( 5 - 7 nm in diameter) are now believed to be a polymerized, !ilamentous form of actin on the basis of arrowhead formation with heavy neromyosin in glycerinated cells. Bundles of microfilaments were usually present in :he myoid and calyceal processes of catfish photoreceptors (also see Burnside, [976), but no consistent relationship was apparent between these bundles and the :hick filaments. Any actin within the thick filaments would represent an alternate !orm aggregated in elevated Mg 2+. Actin aggregation in elevated Mg 2+ in vitro has 3een reported (Cooke et al., 1976), but the resulting paracrystals and F-actin 3undles did not appear morphologically similar to the thick filaments. It is probable :hat the fuzzy filamentous material adhering to and interspersed between synaptic lesicles (Figs. 1--5) represents an aggregated protein system that includes actin
qgs. 11 and 12 are low magnification micrographs of the outer nuclear and outer plexiform layers. :n, cone nuclei; rn, rod nuclei; pt, photoreceptor terminal. ~ig. 11. Nuclear regions following incubation in standard Ringer's solution (Condition A). The mclei in the two layers are virtually indistinguishable, x 7500. qg. 12. Nuclear regions following incubation in high Mg2+, EGTA Ringer's solution Condition C). The outer (cone) layer of nuclei have condensed chromatin, whereas in the inner rod) nuclei the chromatin is dispersed, x 7500.
320
RYAN and POTTER
(LeBeux and Willemot, 1975; Puszkin et al., 1976). The thick filaments appeared in intimate association with this material and seemed to be morphologically continuous with it, as were the synaptic densities and ribbons. The role of thick filaments or their antecendents in the photoreceptor cell is as yet unknown. It seems noteworthy, however, that their presence appeared to be confined to the terminal and myoid. Both these areas perform roles that could conceivably involve the interaction of filamentous arrays for dynamic structural operations. Thus the myoid contracts to change the relative position of the outer segment in response to fluctuating levels of light intensity. The size and structural complexity of the receptor cell terminal may necessitate the use of filamentous systems both for preserving its structural integrity and for participating in a cycle of exocytosis and endocytosis in transmitter release. It is probable that interactions between various populations of filamentous proteins are involved in these dynamic processes. The Mg2*-induced thick filaments described here may represent or arise from such dynamic populations. The phenomenon of chromatin condensation in the outer layer of receptor cell nuclei after incubation in high Mg2§ Ringer's solutions may also be related to the state of aggregation of filamentous proteins within the nucleus. Lestourgeon et al. (1975) have isolated two non-histone proteins from the Myxomycete Physarum and identified them as actin and myosin. They suggest that these contractile proteins may be involved in the process of heterochromatization. They state that the role of Mg 2§ in this event is unclear, although it may be required for actomyosin binding proteins to inhibit the Ca2§ ATPase activity of the actomyosin. Other investigators have suggested that ATP may cause the decondensation of chromatin by acting as a Mg2§ chelating agent (Leake et al., 1972; Comings and Okada, 1976). These results, together with those reported here, suggest a role for filamentous proteins in the condensation of nuclear chromatin. It can be further proposed that this interaction is somehow mediated by Mg2§ ions. We are, however, unable to offer an explanation for the apparent confinement of this phenomenon to the outer layer of nuclei. Because the two layers are believed to segregate into rod and cone nuclei and because it is the cones (outer layer) that are most active in the light-adapted state, we attempted to reverse the condensation by using dark-adapted retinae. No correlation with the ~tate of adaptation was found, since under these conditions, condensed chromatin was still confined to the outer layer of nuclei.
Acknowledgements This investigation was supported by National Institutes of Health grant NS 08964. The authors gratefully acknowledge Dr Gary Hafner's careful review of the manuscript and the expert technical assistance provided by Ms Betsy Osborne and Ms Kitty Johansen.
Mg2+-dependent changes in catfish photoreceptors
321
References BRAY, D. and THOMAS, C. (1976) Unpolymerized actin in tissue cells. In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 3 (edited by GOLDMAN, R., POLLARD, T., and ROSENBAUM, J.), pp. 461--73. BURNSIDE, B. (1976) Microtubules and actin filaments in teleost visual cone elongation and contraction. Journal of Supramolecular Structure 5 , 2 5 7 - 7 5 . COMINGS, D.E. and OKADA, T.A. (1976) Fine structure of the heterochromatin of the kangaroo rat Dipidomys ordii, and examination of the possible role of actin and myosin in heterochromatin condensation. Journal of Cell Science 2 1 , 4 6 5 - 7 7 . COOKE, R., CLARKE, M., yon WEDEL, R. J. and SPUDICH, J. A. (1976) Supramolecular forms of Dictyostelium actin. In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 3 (edited by GOLDMAN, R., POLLARD, T., and ROSENBAUM, J.), pp. 5 7 5 - 8 7 . KARNOVSKY, M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell Biology 27, 1 3 7 A - 1 3 8 A . KARNOVSKY, M.J. (1967) The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. Journal of Cell Biology 3 5 , 2 1 3 - 3 6 . LEAKE, R. E., TRENCH, M. E. and BARRY, J. M. (1972) Effect of cations on the condensation of hen erythrocyte nuclei and its relation to gene activation. Experimental Cell Research 71, 17-26. LEBEUX, Y. J. and WILLEMOT, J. (1975) An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. II. The synapses. Cell and Tissue Research 160, 37--68. LESTOURGEON, W.M., FORER, A., YANG, Y., BERTRAM, J. S. and RUSCH, H. P. (1975) Contractile proteins: Major components of nuclear and chromosome non-histone proteins. Biochimica et Biophysica Acta, 379, 5 2 9 - 5 2 . NIEDERMAN, R. and POLLARD, T. D. (1975) Human platelet myosin. II. In vitro assembly and structure of myosin filaments. Journal of Cell Biology 67, 7 9 - 9 2 . PUSZKIN, S., KOCHWA, S. and ROSENFIELD, R.E. (1976) Ultrastructural evidence of association of contractile proteins in synaptosomes. In: Cold Spring Harbor Conferences on Cell Proliferation, Vol. 3 (edited by GOLDMAN, R., POLLARD, T. and ROSENBAUM, J.), pp. 6 6 5 - 7 0 . ROSENBLUTH, J. (1971a) Myosin-like aggregates in trypsin-treated smooth muscle cells. Journal of Cell Biology 48, 1 7 4 - 8 8 . ROSENBLUTH, J. (1971b) Myosin-like tactoids in trypsin-treated blood platelets. Journal of Cell Biology 50, 9 0 0 - 4 . SCHOENBERG, C. F. (1969) An electron microscope study of the influence of divalent ions on myosin filament formation in chicken gizzard extracts and homogenates. Tissue and Cell 1, 83-96. STOSSEL, T. P. and POLLARD, T. D. (1973) Myosin in polymorphonuclear leukocytes. Journal of Biological Chemistry 248, 8 2 8 8 - 9 4 .
