Search PubMed⌕ Search

Biomedical subjects

D S Papermaster

Publications and source records attributed to D S Papermaster.

At least 55 records · Page 3Linked to original sources
PubMed1984

Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy.

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA.

Acrylic Resins↗
PubMed1984

Preembedding labeling with biotinylated antibodies and subsequent visualization of the biotin groups exposed on thin sections.

The feasibility of labeling cell membranes with biotinylated ligands and detecting the biotin groups on thin sections was investigated. Fixed retinal tissue was incubated with biotinyl- antiopsin . Half of the biotinyl-antibody labeled retinal tissue was incubated with avidin-ferritin (AvF) and embedded in Epon (preembedding reaction). The second half was embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Thin sections of this preparation were incubated with AvF to detect biotinyl-antibodies exposed by the sectioning (postembedding reaction). Biotin groups on the thin section surface could be readily visualized with AvF. Stereoscopic images demonstrated that the ferritin particles were localized only on the exposed surface of the thin section. The labeling was highly specific, with a very low background. Quantitative analysis was employed in order to determine the optimal reaction conditions for maximizing the labeling density with minimizing nonspecific binding. The possibility of using biotinylated molecules in the study of dynamic cellular events and for the subsequent intracellular localization of biotin on thin sections is suggested.

Animals↗
PubMed1983

Fine structure of a periciliary ridge complex of frog retinal rod cells revealed by ultrahigh resolution scanning electron microscopy.

We studied the junctional region between rod inner segments (RIS) and outer segments (ROS) in frog retinas by high resolution scanning electron microscopy (SEM). Retinas of dark adapted or light exposed Rana pipiens were critical-point-dried and RIS and ROS were split and coated with ultrathin metal films of niobium and chromium--or decorated with gold--and imaged in a new SE-I imaging mode. The connecting cilium (CC) usually broke at the base of the RIS and remained attached to the ROS. The outer part of the CC plasmalemma expanded to form liplike protrusions beyond which disks evaginated with successively larger diameter until they reached the full width of the ROS. The CC rose out from an invagination of the RIS apical plasma membrane (PM). On the lateral walls of this invagination, a highly ordered complex of nine symmetrically arrayed ridges and grooves rose steeply and extended laterally approximately 0.4-1 micron on the adjacent RIS PM. On the apical plasmalemma, the ridges and grooves formed groups of three to four parallel rows that surrounded the invagination. The grooves were bridged by filaments anchored at the top edges of the ridges. This highly ordered structure we term the periciliary ridge complex (PRC). Its ninefold symmetry apparently reflects the 9 + 0 microtubule organization of the CC axoneme. The three-dimensional structure revealed by SEM was confirmed by transmission electron microscopy (TEM) of sections of Epon-embedded retinas. TEM-immunocytochemistry on thin sections of retinas embedded in glutaraldehyde cross-linked albumin suggested that the PRC and the CC may participate in opsin transport and disk morphogenesis.

Animals↗
PubMed1983

Differential distribution of opsin in the plasma membrane of frog photoreceptors: an immunocytochemical study.

Opsin molecules on the surface of frog photoreceptors were visualized by immunocytochemistry at the ultrastructural level. Isolated retinas were immersed in biotinyl-antibody to bovine opsin followed by avidin-ferritin conjugates. Anti-opsin bound to the plasma membrane and to the surface of the most basal discs of red rod outer segments. Inner segment plasma membranes of red rod photoreceptors were devoid of anti-opsin label except for the apical plasma membrane in the region of the recently described periciliary ridge complex. The connecting cilium surface from its base at the periciliary region to the site of new disc evagination was almost free of anti-opsin binding, an observation in consonance with prior studies of thin sectioned retinas embedded in glutaraldehyde cross-linked bovine serum albumin. These results indicate that the continuous plasma membrane of photoreceptors is highly polarized. Opsin, which is free to diffuse throughout the outer segment plasma membrane and along the discs, does not back-diffuse onto the inner segment plasma membrane. The periciliary ridge complex and the base of the connecting cilium are possible sites of restriction of opsin mobility. This study also has provided new insight into the molecular structure of frog visual pigments. Frog green rod and cone outer and inner segment plasma membranes were not labeled by this sheep antiserum to bovine opsin. In contrast, discs of green ROS and the lamellae of some cones were labeled when these antibodies were applied to albumin embedded thin sections of frog retinas. Apparently, only internal or intramembraneous domains of green ROS and cone visual pigments were recognized by this antibody while both internal and extracellular domain(s) of red ROS opsin were reactive.

Animals↗
PubMed1982

Cone lamellae and red and green rod outer segment disks contain a large intrinsic membrane protein on their margins: an ultrastructural immunocytochemical study of frog retinas.

In addition to rhodopsin, the disk membranes of rod outer segments (ROS) contain a large integral membrane protein (mol. wt 290,000). This protein was previously localized by immunocytochemistry to the margins and incisures of disks in frog red ROS by specific antibody applied to thin sections of bovine serum albumin embedded retinas (Papermaster et al., 1978b, J. Cell Biol. 78, 415-425). Upon further study of the reactions of this antibody with outer segments of other photoreceptor classes in frog retina, labeling of the short incisures and margins of green ROS and margins of cone outer segment lamellae is also observed. Thus the large protein participates in the structure of the edges of disks and lamellae of all photoreceptors in the frog. In addition, labeling of the inter-incisure surface of all photoreceptor classes was observed at high antibody concentration. In order to interpret this labeling, the effect of dilution on labeling density was determined and double reciprocal plots (Markham and Benton, 1931, J. Am. Chem. Soc. 53, 497) were employed to evaluate the relative affinity and heterogeneity. There was considerable deviation from linearity in the plots of labeling disk interiors compared to the relatively linear plots of disk incisure labeling which suggests that the interior sites contain a weakly cross-reacting antigen or that the serum contains a lower concentration of antibody weakly reactive with another antigen.

Animals↗
PubMed1979

Messenger RNA of opsin from bovine retina: isolation and partial sequence of the in vitro translation product.

Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH(2) terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH(2)-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids (Met, Asn, Pro, Phe, Tyr, Val) provided the following result: [Formula: see text] (X is unknown). This partial sequence of the cell-free product corresponds exactly to the published NH(2)-terminal segment of native opsin (21 residues long) and extends beyond this region. Met-1 was shown to be the initiator methionine residue, because only the initiator [(35)S]Met-tRNA(1) (Met)-not the internal [(35)S]Met-tRNA(2) (Met)-donated the NH(2)-terminal methionine. This finding essentially rules out the possibility that Met-1 was preceded by a peptide that was rapidly cleaved. Thus opsin, and not a precursor, is the immediate product of opsin mRNA translation.

Amino Acid Sequence↗
PubMed1978

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas.

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.

Animals↗
PubMed1978

Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.

Immunocytochemical techniques have localized a large protein which is an intrinsic membrane component of isolated frog rod outer segments (ROS). This large protein whose apparent mol wt is 290,000 daltons comprises about 1--3% of the ROS membrane mass. Its molar ratio to opsin is between 1:300 and 1:900. Adequate immune responses were obtained with less than 30 microgram (100 pmol) of antigen per rabbit. Antibodies to the large protein were used for its localization on thin sections of frog retina embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specifically bound antibodies were detected by an indirect sequence with ferritin-conjugated antibodies. This technique detected the protein which is represented by 1,000--3,000 molecules per disk. This indicates that the procedure is sufficiently sensitive for analysis of membrane components in low molar proportions. The large protein was specifically localized to the incisures of ROS disks which divide the disks into lobes and to the disk margin. Thus, opsin is mobile within the membrane of the disk while the large protein is apparently constrained to the disk edges. This finding raises the possibility that special functions are also localized ot his unusual region of high curvature, and that collisions of bleached opsin with these edges are physiologically important in couter segment function.

Animals↗