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Cover image for Vol. 15 Issue 5

Edited By: Michael S. Marks, Mark C. P. Marsh, Trina A. Schroer, Tom H. Stevens

Online ISSN: 1600-0854

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  • AP180 Couples Protein Retrieval to Clathrin-Mediated Endocytosis of Synaptic Vesicles

    AP180 Couples Protein Retrieval to Clathrin-Mediated Endocytosis of Synaptic Vesicles

    Independent diffusion ofSVproteins following nerve stimulation is sustained for long periods followingLAPinactivation. Third instar larval NMJ preparations from the indicated genotypes were dissected, incubated with FlAsH-EDT2 and muscle 4 from segment A2 or A3 was exposed to 488-nm-filtered fluorescent light for 5 min. Clap4C; lap = Clap4C; lap1/lapsd3. Following illumination, the larval NMJs were stimulated continuously with high K+ (90 mm) Jan's solution for 5 min (A and B) or 30 min (C and D) or 10 min (E and F). Following nerve stimulation, the larvae were washed rapidly with Ca2+-free HL-3 saline and fixed. The NMJs were then stained for a pair of SV proteins Syn and vGlut or Syt1 and CSP. Panels A–D also show HRP-stained NMJs (blue) to mark the neuronal membrane. The stimulation paradigm does not affect SV protein colocalization in wild-type control larval NMJs in which most SV proteins are colocalized although we note that not all SV proteins are fully colocalized even in the wild-type larval NMJs. In contrast, there are more punctate in LAP-inactivated synaptic boutons and SV proteins are often not colocalized in these punctate (arrows). G and H) Following 10 min of stimulation with high K+, nerve terminals were allowed to rest in Ca2+-free HL-3 saline for an additional 20 min prior to fixation. Following the resting period, dramatically large punctate can be observed positive for both Syt1 and Syn (arrowheads) in larval NMJs whose LAP is inactivated, while some portions of the punctate within the bouton are positive for only one of the two SV proteins (arrows). The protocol for both control and experimental samples was identical. Scale bars = 10 µm.

  • Nonmuscle Myosin II Is a Critical Regulator of Clathrin-Mediated Endocytosis

    Nonmuscle Myosin II Is a Critical Regulator of Clathrin-Mediated Endocytosis

    MIIBis closely associated with coated pits in the Wt and clathrin-coated pit (CCP) structure is abnormal inMIIB KOcells. A) Immunogold labeling for MIIB in Wt cells. Particles are circled in red for better visibility. Gold particles were identified in anaglyphs. From two separate preparations: On left, 18 nm gold, on right, 12 nm gold. Bar = 90 nm. B) Comparison of highly-invaginated pits in thin section (left) and in rotary shadowed replicas (right arrows indicate coated pit). Far right panel is an anaglyph. C) The same comparison in MIIB KO cells. Note that the coated pits are distorted. Bar = 150 nm. D) CCP profiles in thin sections from Wt cells. S = shallow, C = curved, I = invaginated, CCV = clathrin coated vesicle. E) CCP profiles in MIIB KO cells. Arrowheads indicate distorted pits. Bar = 100 nm. F) Distribution of CCP profiles in Wt cells as a percentage of total (N = 142 from 20 cells) observed. G) Distribution of CCP profiles (N = 132 from 15 cells) in MIIB KO cells as compared to Wt. The frequency of shallow and curved coated pits is increased, whereas the frequency of CCVs is greatly decreased. H) Blebbistatin treatment results in a greater frequency of shallow and curved coated pits compared to untreated fibroblasts. The frequency of CCV's is decreased. Untreated Ct, N = 138 from 18 cells, bleb treated, N = 144 from 16 cells. I) Shallow coated pits measured in rotary shadowed replicas from unroofed fibroblasts are larger in diameter in MIIB KO cells (t-test, p < 0.001, N = 20 each). The same result was obtained from measurements made from thin sections, except that the diameters for shallow coated pits in both Wt and MIIB KO cells were approximately 30% less than in replicas. J) Histograms showing the distribution of coated pit symmetry (width in x divided by width in y) measured in rotary shadowed preparations. Coated pits are more asymmetrical in MIIB KO cells.

  • A Unique C-terminal Domain Allows Retention of Matrix Metalloproteinase-27 in the Endoplasmic Reticulum

    A Unique C-terminal Domain Allows Retention of Matrix Metalloproteinase-27 in the Endoplasmic Reticulum

    Predicted domain structure of humanMMP-27 and comparison of C-terminal domains betweenMMP-27 andMT-MMPs. A) Domain structure of MMP-27 was deduced from sequence comparison with other MMPs (see details in text). By analogy, the hemopexin-like domain is shaped like a four-bladed beta-propeller closed by a disulfide bond between Cys279 and Cys465 (C) residues. B) Sequence alignments. Primary sequences of carboxy-terminal extensions of the four human transmembrane MT-MMPs (MMP-14, -15, -16, -24) were compared to that of MMP-27 using ClustalX2. For clarity, all selected sequences start with the Cys ending the hemopexin domain; numbering refers to MMP-27 sequence. Hydrophobic residues corresponding to the transmembrane domain in MMP-14, -15, -16 and -24 are boxed, the potential transmembrane domain of MMP-27 is underlined and its charged residues are in bold. All domain annotations are from Uniprot (MMP-27 = Q9H306; MMP-14 = P50281; MMP-15 = P51511; MMP-16 = P51512; MMP-24 = Q9Y5R2), except for the prediction of the potential transmembrane domain of MMP-27 (Spoctopus).

  • CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum

    CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum

    CNIH4promotes theERexit ofHA-β2AR-A7and interacts with Sec23 and Sec24. A) HEK293 cells were transfected with β2AR-hRluc or β2AR-A7-hRluc along with calnexin-vYFP in the presence or absence of CNIH4 and BRET signals were measured. Data shown are the mean ± SEM of four independent experiments performed at comparable level of total fluorescence and luminescence for each condition. Statistical significance of the difference was assessed by paired Student's t-test using the Prism software. B) Endogenous Sec23 or Sec24 isoforms (B or D) were immunoprecipitated from lysates of HEK293 cells expressing vYFP-CNIH4 (L or S) or calnexin-vYFP using specific anti-Sec antibody. The presence of CNIH4 or calnexin was detected by western blot analysis using an anti-GFP antibody. The results shown are representative of three independent experiments.

  • The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network

    The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network

    The multiple features of thePlasmodium bergheiliver stage tubovesicular network (LS-TVN). A) Live confocal imaging of liver stage P. berghei expressing UIS4-mCherry and cytoplasmic GFP reveals different morphologies of the LS-TVN: elongated membrane clusters (left), vesicles in the host cell cytoplasm (center), and a thin tubule protruding from the PVM (right). Live imaging was performed 20 h after infection of hepatoma cells. Features are marked with white arrowheads. B) The UIS4- and IBIS1-positive tubules rapidly extend from and retract to the PVM. Confocal time-lapse imaging of a UIS4-mCherry-expressing parasite at 22 h (top) and an IBIS1-mCherry-expressing parasite at 26 h (bottom) after infection of hepatoma cells. Tubules are indicated with a yellow arrow, and the time is shown in minutes and seconds. C) Imaging of IBIS1-mCherry-expressing P. berghei reveals similar features of the LS-TVN, i.e. elongated membrane cluster (left), vesicles in the host cell cytoplasm (center), and a membrane tubule (right). Infected cells were fixed 26 h post-infection and imaged by epifluorescence microscopy. Features are indicated with a yellow arrowhead. Scale bars, 10 µm. D) Quantification of the distinct UIS4- and IBIS1-positive membranous structures in fixed infected hepatoma cells at three different time points: 6 h after infection (the period of sporozoite transformation into exo-erythrocytic forms), 24 h after infection (onset of parasite replication and growth), and 48 h after infection (mature liver stages). Values are shown as mean (± SD) from three separate wells (n = 100 per well) of one experiment and are representative of two experimental repetitions.

  • Glycosylation of the Nuclear Pore

    Glycosylation of the Nuclear Pore

    O-GlcNAcmodification integrates metabolic information. The nucleotide-sugar UDP-GlcNAc is produced through the HBP. This pathway integrates metabolic signals from carbohydrate (glucose), amino acid (glutamine), fatty acid (acetyl-CoA) and nucleotide (UTP) metabolism. Because ATP is required for UTP biosynthesis, the HBP also reports on energy charge. UDP-GlcNAc is required for various forms of glycosylation, including the production of the O-GlcNAc modification. O-GlcNAc is produced by the enzyme OGT and removed from proteins by the enzyme OGA. Sites of O-GlcNAc modification are often competitively phosphorylated by kinases.

  • AP180 Couples Protein Retrieval to Clathrin-Mediated Endocytosis of Synaptic Vesicles
  • Nonmuscle Myosin II Is a Critical Regulator of Clathrin-Mediated Endocytosis
  • A Unique C-terminal Domain Allows Retention of Matrix Metalloproteinase-27 in the Endoplasmic Reticulum
  • CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum
  • The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network
  • Glycosylation of the Nuclear Pore

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