David D. Sabatini

Bio: David D. Sabatini is an academic researcher from New York University. The author has contributed to research in topics: Endoplasmic reticulum & Golgi apparatus. The author has an hindex of 59, co-authored 119 publications receiving 17814 citations. Previous affiliations of David D. Sabatini include Rockefeller University & Yale University.

Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.

Abstract: The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.

Polarized monolayers formed by epithelial cells on a permeable and translucent support.

Abstract: An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.

Mechanisms for the incorporation of proteins in membranes and organelles.

Abstract: Because of the high degree of organizational complexity of eucaryotic cells, it is clear that the implementation of their genetic programs must, in many cases, involve a complex sequence of cotranslational and posttranslational events that are necessary to transfer polypeptides from their sites of synthesis to their sites of function. It is difficult to envisage the existence of a single general mechanism that would ensure that all polypeptides released from ribosomes attain their correct subcellular destination . This is because there are, in a eucaryotic cell, at least as many possible destinations for a newly synthesized polypeptide as there are different compartments and membrane systems. Moreover, it is not likely that completed and fully folded polypeptides, with their charged and polar residues exposed to the aqueous environment, could freely traverse hydrophobic barriers constituted by phospholipid bilayers, which are the universal feature of all cell membranes (208). Instead, it may be expected that special mechanisms have evolved that direct polypeptides to specific membranes and, when necessary, assist them in their passage across the hydrophobic barriers . These mechanisms must involve specific receptors for structural features ofthe polypeptides and may entail conformational changes or even extensive structural modifications of the polypeptide, as well as the expenditure of energy . In this paper we consider mechanisms for the transfer of newly synthesized polypeptides to their sites of function in different subcellular membranes and organelles, and discuss models in which specific features ofthe polypeptides serve as signals to direct them along selected subcellular pathways to their final destination . These signals may act during translation or after synthesis of the polypeptide is completed and may or may not be removed from the initial product of translation . Transient or permanent signals within polypeptides destined to membranes would also account for the final characteristic orientation of membrane proteins with respect to the phospholipid bilayer . Several other reviews discussing various aspects of this subject have recently appeared (15, 44, 53, 117a, 127, 243a) .

Positional cloning of the mouse obese gene and its human homologue

Abstract: The mechanisms that balance food intake and energy expenditure determine who will be obese and who will be lean. One of the molecules that regulates energy balance in the mouse is the obese (ob) gene. Mutation of ob results in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.

The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique.

Abstract: The early stages of absorption of intravenously injected horseradish peroxidase in proximal tubules of mouse kidney were studied with a new ultrastructural cytochemical technique. In animals killed as early as 90 sec after injection, reaction product was found on the brushborder membranes and in the apical tubular invaginations. From the latter structures it was transported to the apical vacuoles, in which it was progressively concentrated to form protein absorption droplets. The method, which employs 3,3'-diaminobenzidine as oxidizable substrate, gives sharp localization and is sensitive. This system is advantageous in studying the early stages of renal tubular protein absorption, since small amounts of protein on membranes and in tubules and vesicles can be detected easily. The method also appears promising for studying protein transport in a variety of other cells and tissues.

Signal integration in the endoplasmic reticulum unfolded protein response

Abstract: The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways - cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.