Pars plana vitrectomy, subretinal injection of tissue plasminogen activator, and fluid-gas exchange for displacement of thick submacular hemorrhage in age-related macular degeneration.

PURPOSE: To evaluate a new procedure for displacement of large, thick submacular hemorrhage in patients with age-related macular degeneration. METHODS: Retrospective review of 11 eyes of 11 patients with age-related macular degeneration and thick submacular hemorrhage (defined as causing retinal elevation detectable on stereo fundus photographs) treated with vitrectomy, subretinal injection of tissue plasminogen activator (25 or 50 microg), and fluid-gas exchange with postoperative prone positioning. Outcome measures included displacement of hemorrhage from the fovea, best postoperative visual acuity, and final postoperative visual acuity. RESULTS: In the 11 affected eyes of 11 patients (seven men and four women; mean age, 76 years), preoperative visual acuity ranged from 20/200 to hand motions. With surgery, subretinal hemorrhage was displaced from the fovea in all 11 cases. Mean postoperative follow-up was 6.5 months (range, 1 to 15 months). Best postoperative visual acuity varied from 20/30 to 5/200, with improvement in nine (82%) cases and no change in two cases. Eight eyes (73%) measured 20/200 or better, with four of these eyes (36%) 20/80 or better. Final postoperative visual acuity ranged from 20/70 to light perception, with improvement in eight (73%) cases, no change in one case, and worsening in two cases. A statistically significant difference was found between preoperative and best postoperative visual acuity (P =.004) but not between preoperative and final visual acuity (P =.16). Hemorrhage recurred in three (27%) eyes, causing severe visual loss in one eye. CONCLUSIONS: This technique displaces submacular hemorrhage from the fovea and can improve vision in patients with age-related macular degeneration. However, recurrence of hemorrhage occurred in 27% of eyes and caused severe visual loss in one eye. A randomized, prospective clinical trial is necessary to determine the efficacy of this technique in comparison with other proposed treatments.

Kinesin and cytoplasmic dynein binding to brain microsomes.

Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.

Kinectin, a major kinesin-binding protein on ER.

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing. mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.

Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein.

Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.

Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes.

We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.

Localization of cytoplasmic dynein to mitotic spindles and kinetochores.

What is the origin of the forces generating chromosome and spindle movements in mitosis? Both microtubule dynamics and microtubule-dependent motors have been proposed as the source of these motor forces. Cytoplasmic dynein and kinesin are two soluble proteins that power membranous organelle movements on microtubules. Kinesin directs movement of organelles to the 'plus' end of microtubules, and is found at the mitotic spindle in sea urchin embryos, but not in mammalian cells. Cytoplasmic dynein translocates organelles to the 'minus' end of microtubules, and is composed of two heavy chains and several light chains. We report here that monoclonal antibodies to two of these subunits and to another polypeptide that associates with dynein localize the protein to the mitotic spindle and to the kinetochores of isolated chromosomes, suggesting that cytoplasmic dynein is important in powering movements of the spindle and chromosomes in dividing cells.

The mechanism and regulation of fast axonal transport.

Recent in vitro studies of microtubule-dependent organelle movement have provided a great deal of information on the molecular mechanism of fast axonal transport. Microtubule-dependent organelle movement occurs in most cells, but in neurons active transport is absolutely necessary for materials to travel from the cell body to the synapse. Since fast transport is crucial for neuronal survival, it is likely that specialized regulatory mechanisms have been developed. It is clear that the microtubule-based motors, kinesin and cytoplasmic dynein are the enzymes that power organelle motility; however, additional cytoplasmic components are required to create an 'organelle translocation complex' that is competent for transport. Organelle transport might be regulated at the level of any of these components, i.e. the motors, their accessory factors, or the organelle binding sites. The direction of organelle movement is probably governed by the membrane binding site. In this review we discuss these topics and consider the mechanism of transport of the retrograde motor, cytoplasmic dynein, to the nerve terminal, and possible ways that unidirectional transport could occur on the non-polarized array of microtubules found in some dendrites.

Cytoplasmic dynein is a minus end-directed motor for membranous organelles.

The role of cytoplasmic dynein in microtubule-based organelle transport was examined using a reconstituted assay developed from chick embryo fibroblasts. Factors present in a high-speed cytosol caused the movement of purified organelles on microtubules predominantly in the minus end direction. Inactivation of cytoplasmic dynein in the high-speed cytosol by vanadate-mediated UV photocleavage inhibited minus end-directed organelle motility by over 90%. Addition of purified cytoplasmic dynein to the inactive cytosol restored minus end-directed organelle motility, although purified cytoplasmic dynein by itself did not support organelle movement. We propose that cytoplasmic dynein is the motor for minus end-directed organelle movement, but that additional cytosolic factors are also required to produce organelle motility.