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J M McCaffery

Publications and source records attributed to J M McCaffery.

53 records · Page 3Linked to original sources
PubMed1996

A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15.

A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.

Actins↗
PubMed1996

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.

Acetylation↗
PubMed1996

Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant.

In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.

Amino Acid Sequence↗
PubMed1996

Cell biology of the primitive eukaryote Giardia lamblia.

Giardia lamblia is an extremely primitive or early-diverging eukaryote that has been considered to have no typical ER or Golgi apparatus, although it is a complex and highly developed cell. Both the trophozoite and cyst have unusual surface proteins that enable these stages to survive in very different and hostile environments. We found that G. lamblia forms novel encystation-specific secretory vesicles and can sort cyst wall proteins to a regulated secretory pathway distinct from the constitutive pathway used to transport the variable cysteine-rich protein to the trophozoite surface. Our studies, utilizing novel ultrastructural methods that preserve the endomembranes, as well as IEM, support the idea that G. lamblia has many of the endomembrane protein transport elements and sorting functions of higher cells and that these appeared very early in the evolution of eukaryotic cells.

Animals↗
PubMed1995

Reconstitution of vesiculated Golgi membranes into stacks of cisternae: requirement of NSF in stack formation.

We have developed an in vitro system to study the biochemical events in the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae. The Golgi complex in intact normal rat kidney cells (NRK) is vesiculated by treatment with IQ. The cells are washed to remove the drug and then permeabilized by a rapid freeze-thaw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires ATP and a high speed supernatant from cell extract (cytosol), as revealed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and contain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to promote fusion of transport vesicles with the target membrane in the exocytic and endocytic pathways. Immunoelectron microscopy using Golgi specific anti-mannosidase II antibody reveals that VGMs undergo sequential changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.

Adenosine Triphosphate↗
PubMed1994

Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum.

Newly synthesized proteins are believed to move from the endoplasmic reticulum (ER) to the Golgi by bulk flow, and sorting is assumed to occur exclusively in the trans-Golgi network (TGN). Using quantitative immunoelectron microscopy, we demonstrate that vesicular stomatitis virus glycoprotein (VSV-G) is sorted from resident ER proteins and concentrated 5- to 10-fold in 40-80 nm vesicles during vesicle budding from the ER. Accumulation of VSV-G in pre-Golgi vesicular carriers is the only detectable concentration step in its transport to the TGN. From these results, it is apparent that export from the ER is not exclusively mediated by bulk flow. The ER exerts an unanticipated level of control to insure selective and efficient entry of mature protein into the secretory pathway.

Animals↗
PubMed1994

Giardia lamblia: ultrastructural basis of protein transport during growth and encystation.

Giardia, an early diverging eukaryote, is reported to have no Golgi apparatus. Moreover, the structural basis for its ability to sort key proteins and transport them to the trophozoite plasma membrane or to the extracellular wall during encystation is not well-understood. Therefore, we have used ultrastructural techniques that enhance the endomembrane system to evaluate the presence and relationships of cytoplasmic organelles and structures that correspond to those present in higher cells. In addition to the perinuclear cisternae, we found rough endoplasmic reticulum (ER), transitional elements, putative tubular-vesicular elements, Golgi-like smooth perinuclear membrane stacks, and lysosome-like peripheral vesicles. Moreover, we observed many small (50-80 nm) vesicles, many of which were coated, that resemble the small transport vesicles that carry proteins between successive ER and Golgi compartments. Importantly, many of these membrane elements appeared to be captured in the process of budding (or fusing). These elements of the endomembrane system are present during both vegetative growth and encystation of Giardia lamblia. In contrast, the encystation-specific vesicles (ESV) are novel large regulated secretory vesicles that transport cyst antigens to the nascent wall. The present studies suggest that ESV may have unusual pathways of formation and traffic. Our findings support the idea that Giardia, a primitive parasite, has complex structures for protein transport. The elements that show similarities to higher cells may have evolved early, while those that differ may represent biologic fossils or specializations for the parasitic life cycle.

Animals↗
PubMed1994

Giardia lamblia: traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching.

Both trophozoites and cysts of Giardia lamblia have unique outer surfaces that protect them from very different hostile environments. However, little is known about the transport of these important molecules to the cell surface. We used monospecific anti-recombinant TSA 417 antibodies and mAb 8C5 in double label immunoelectron microscopy to compare the localization and transport of this major trophozoite surface antigen (TSA) with that of a prominent cyst wall epitope during vegetative growth, encystation, and antigenic switching in vitro. TSA 417 is a marker of the constitutive transport of the major plasma membrane protein, while the 8C5 epitope traces a differentiation-regulated secretory pathway to the cyst wall. Both proteins localized to the nuclear envelope endoplasmic reticulum (ER) cisternae, ER, and cytoplasmic membrane cisternae, reflecting their site of synthesis, but only the differentiation-specific epitope 8C5 localized to the encystation-specific vesicles (ESV). These large secretory vesicles form only during encystation and transport cyst antigens to the nascent wall. In contrast, only TSA 417 was found on the outer surface of the plasmalemma of trophozoites and encysting cells and underlaying the walls of many cysts, while only 8C5 localized to the cyst wall. As encystation progressed, TSA 417 disappeared from the plasmalemma and increased in the lysosome-like PV and other large cytoplasmic vesicles. In contrast to their segregation in the ESV and on the cell surface, both TSA 417 and 8C5 were found in the peripheral vesicles, presumably an endocytic compartment. We propose that this may be the initiation of a stage in differentiation-driven antigenic switching of TSA 417, in which this antigen is no longer synthesized or exported to the plasmalemma, but is taken back inside the cell.

Animals↗
PubMed1994

Rab1 and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport.

Members of the rab/YPT1/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocytic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rab1a mutant, rab1a(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rab1 and beta-COP, a subunit of the coatomer complex. Similar to previous observations (Balch et al. 1994. Cell. 76:841-852), VSV-G was concentrated nearly 5-10-fold in vesicular carriers that accumulate in the presence of the rab1a(N124I) mutant. VSV-G containing vesicles and vesicular-tubular clusters were also found to accumulate in the presence of a rab1a effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca2+. These results suggest that the combined action of a Ca(2+)-dependent protein and conformational changes associated with the GTPase cycle of rab1 are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.

Animals↗
PubMed1993

Reduction of postoperative against-the-rule astigmatism by lateral incision technique.

A group of 80 preoperative against-the-rule (AR) eyes had cataract removal and intraocular lens implantation with lateral (temporal) incision and suturing. Fifty-six of the 80 eyes had extracapsular cataract extraction (ECCE) and 24 had phacoemulsification (KPE) with capsulorhexis. For both procedures, subgroups (21 of ECCE, ten of KPE) with high preoperative AR astigmatism (> or = -2.0 D) were evaluated. Analysis of the postoperative data provided the following: (1) induced postoperative cylinder decrease, (2) effect of cutting and not cutting sutures, (3) induced axial shift, (4) postoperative astigmatic change as a function of time, and (5) comparative effectiveness of ECCE and KPE lateral incision procedures in reducing postoperative AR astigmatism.

Astigmatism↗
PubMed1991

Detection of individual Peyer's patch T cells that produce interleukin-5 and interferon-gamma.

A method was developed to detect individual interferon-gamma (IFN-gamma)- and interleukin-5 (IL-5)-producing cells among freshly isolated T cell populations and long term lines of CD4+ Peyer's patch T cells using frozen semi-thin sections of paraformaldehyde fixed-T cells and immunofluorescence techniques. Using this method, individual CD4+ Peyer's patch T cells could be shown to produce IFN-gamma, characteristic of the T helper 1 (Th1) T cell type, IL-5, characteristic of the T helper 2 (Th2) T cell type, as well as both IFN-gamma and IL-5. These data support the notion that Th1 and Th2 cells derive from a common T cell precursor.

Animals↗
PubMed1986

A review of 584 3M style 30 intraocular lens implants.

The 3M style 30 intraocular lens was introduced in January 1980 as a closed-loop, uniplanar posterior chamber lens intended for in-the-bag implantation. Between February 13, 1980, and February 28, 1982, 584 of these lenses were implanted by one of us. Except for a few lost to follow-up, these patients have now had a minimum of two years of follow-up under our care. Using a microcomputer-based data-base management system, we have generated graphic and tabular presentations of follow-up, age at time of surgery, best postoperative visual acuity, preoperative and postoperative pathology, and surgical events and complications. A brief discussion of the advantages of the in-the-bag location and the style 30 design characteristics is included. We have been able to profile, compare, and correlate numerous preoperative, operative, and postoperative parameters.

Adult↗
PubMedDate not supplied

Ultrastructural effects of lactoferrin binding on Giardia lamblia trophozoites.

Lactoferrin and its derived N-terminal peptide may be important host defenses against Giardia lamblia. We showed earlier that lactoferrin and the derived peptides have potent giardicidal activity in vitro. Using indirect immunofluorescence, we now demonstrate binding of lactoferrin and the peptides to the live trophozoite surface. Iron strongly inhibited binding of lactoferrin, and decreased binding of the peptides, while certain divalent metal ions decreased binding of all forms by about half. Lactoferrin and the peptides caused striking and complex morphologic changes in the trophozoite plasmalemma, endomembranes and cytoskeleton, and increased the electron density of the lysosome-like peripheral vacuoles.

Animals↗