Guanylyl cyclase activating protein. A calcium-sensitive regulator of phototransduction.

Guanylyl cyclase activating protein (GCAP1) has been proposed to act as a calcium-dependent regulator of retinal photoreceptor guanylyl cyclase (GC) activity. Using immunocytochemical and biochemical methods, we show here that GCAP1 is present in rod and cone photoreceptor outer segments where phototransduction occurs. Recombinant and native GCAP1 activate recombinant human retGC (outer segment-specific GC) and endogenous GC(s) in rod outer segment (ROS) membranes at low calcium. In addition, we isolate and clone a retinal homolog, termed GCAP2, that shows approximately 50% identity with GCAP1. Like GCAP1, GCAP2 activates photoreceptor GC in a calcium-dependent manner. Both GCAP1 and GCAP2 presumably act on GCs by a similar mechanism; however, GCAP1 specifically localizes to photoreceptor outer segments, while in these experiments GCAP2 was isolated from extracts of retina but not ROS. These results demonstrate that GCAP1 is an activator of ROS GC, while the finding of a second activator, GCAP2, suggests that a similar mechanism of GC regulation may be present in outer segments, other subcellular compartments of the photoreceptor, or other cell types.

Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts.

Intermediate filaments (IFs) undergo specific rearrangements in cells, some aspects of which can be induced experimentally. Centripetal aggregation of the IF network, for example, can be produced by a variety of perturbations. However, the source of motive force is clear for neither in vivo nor experimentally generated IF movements, since, unlike microtubules and actin filaments, IFs have no known force-generating system directly associated with them. We recently obtained evidence that the drug-induced aggregation of vimentin IFs in fibroblasts is an active event, which requires ATP and involves the actin cytoskeleton. In the present study, we sought to test the hypothesis that IF aggregation is driven by a centripetally directed contraction of the actomyosin cortex. To that end, we have permeabilized fibroblasts with Triton X-100 in a stabilizing buffer and reactivated cytoskeletal movements in vitro, under defined solution conditions. Upon nucleotide treatment, these permeabilized cells undergo a nucleotide-dependent centripetal aggregation of vimentin IFs similar in appearance and time course to that induced in intact cells by drug treatment. During in vitro IF aggregation, the permeabilized cells remain fully spread and adherent to the substratum, and the distal ends of the microtubules and actin microfilaments retain their positions in the cell periphery, IF aggregation is accompanied by a contraction of F-actin and myosin into focal aggregates in the same perinuclear region in which the IFs accumulate. If permeabilized cells are treated with the actin-severing protein gelsolin prior to the reactivation of IF movement, the actin cytoskeleton is eliminated and IF aggregation fails to occur when ATP is added. These results strongly support a model in which the motive force for IF movement is supplied indirectly by association with a contracting actomyosin network.

Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein.

Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and alpha-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.

[Organization of stress fibrils in cultured fibroblasts studied by using an actin filament-fragmenting protein]. / Issledovanie organizatsii stress-fibrill kul'tiviruemykh fibroblastov s pomoshch'iu belka, fragmentiruiushchego aktinovye filamenty.

Earlier we isolated a 1:1 complex of 90 kD-protein and actin from bovine brain. This complex was able to fragment actin filaments. Effects of this complex on the cytoskeleton of mouse and quail embryo fibroblasts are described. The cells were extracted with Triton X-100, and the resulting cytoskeletons were treated with the complex. Tetramethylrhodaminylphalloin and actin antibody staining failed to detect actin in preparations treated with the 90 kD protein-actin complex. Electrophoretic data confirmed actin solubilization upon this treatment. Immunofluorescent microscopy showed that actin solubilization was accompanied by extraction of vinculin and alpha-actinin from focal contacts and stress-fibers. In contrast, myosin distribution in treated cytoskeletons did not differ significantly from that in control extracted cells: in both the cases myosin was found mainly in the stress-fibers. Thus, myosin localization in stress-fibers does not depend on actin and is probably controlled by some other cytoskeletal component(s).

[Study of a protein complex from the brain which reduces actin viscosity. I. Composition and various properties of the complex]. / Issledovanie belkovogo kompleksa iz mozga, ponizhaiushchego viazkost' aktina. I. Sostav i nekotorye svoistva kompleksa.

The method of isolation from bovine brain of a preparation containing 90 kDa- and 42 kDa-proteins is described. This preparation shortens actin filaments and therefore decreases viscosity of F-actin. The 42 kDa-component was identified as actin by one-dimensional peptide mapping. Quantitative densitometry has demonstrated that 90 kDa-protein and actin are present in the preparation in equimolar ratio. Fractionation of the preparation by gel-filtration, analytical centrifugation or electrophoresis under non-denaturing conditions showed that 90 kDa-protein and actin are in a light complex. This complex consists of one actin molecule and one molecule of 90 kDa-protein and has a sedimentation coefficient of 3.5S. Both beta- and gamma-isoelectric forms of actin are present in the complex.

[Study of a protein complex from the brain which reduces actin viscosity. II. The complex inhibits elongation of actin filaments at the end preferable for polymerization and fragments the filaments]. / Issledovanie belkovogo kompleksa iz mozga, ponizhaiushchego viazkost' aktina. II. Kompleks ingibiruet élongatsiiu aktinovykh filamentov na predpochtitel'nom dlia polimerizatsii kontse i fragmentiruet filamenty.

Functional properties of the protein complex from bovine brain that shortens actin filaments are described. In the presence of Ca2+ complex shortens actin filaments and increases the initial rate of actin polymerization. In the absence of free calcium ions the complex loses its accelerating effect on actin polymerization, but still possesses actin filament shortening activity. Neither phalloidin nor tropomyosin prevent the shortening of actin filaments induced by the protein complex. Therefore the protein complex causes the fragmentation of actin filament. The data on actin polymerization in the presence of F-actin nuclei have indicated that the protein complex inhibits the elongation step of actin polymerization. The analysis of elongation in the presence of both the protein complex and cytochalasin D has demonstrated that the inhibition occurs on the fast-growing end of actin filaments.

Phalloidin and tropomyosin do not prevent actin filament shortening by the 90 kD protein-actin complex from brain.

Previously we reported the purification from bovine brain of the 90 kD protein-actin complex that shortens actin filaments. In the present work we study the effect of this complex on actin polymerized in the presence of phalloidin (PL) or tropomyosin (TM) which are known to stabilize actin filaments. The effect of the complex has been compared with that of cytochalasin D (CD), a fungal metabolite that also shortens actin filaments. Low shear viscosimetry and electron microscopy showed that PL or TM could not prevent the shortening of actin filaments in the presence of 90 kD protein-actin complex whereas they effectively protected actin filaments from shortening by CD. We conclude that the 90 kD protein-actin complex is a more potent filament-shortening factor than CD.

[Structural studies on tubulin in flat sheets formed in the presence of zinc]. / Strukturnye issledovaniia tubulina v ploskikh sloiakh, obrazovannykh v prisutstvii tsinka.

Structure of the tubulin molecule was determined from microtubule images by methods of three dimensional reconstruction. We detected two types of zinc-induced sheets with different space latticies. Using the model of the tubulin molecule obtained by three dimensional reconstruction, the relationship of its projections was established by the method of optical filtration. Structural peculiarities in tubulin polymorphism are considered.