Cytoplasmic Capes Are Nuclear Envelope Intrusions That Are Enriched in Endosomal Proteins and Depend upon βH-Spectrin and Annexin B9

It is increasingly recognized that non-erythroid spectrins have roles remote from the plasma membrane, notably in endomembrane trafficking. The large spectrin isoform, βH, partners with Annexin B9 to modulate endosomal processing of internalized proteins. This modulation is focused on the early endosome through multivesicular body steps of endocytic processing and loss of either protein appears to cause a traffic jam before removal of ubiquitin at the multivesicular body. We previously reported that βH/Annexin B9 influenced EGF receptor signaling. While investigating this effect we noticed that mSptiz, the membrane bound precursor of the secreted EGF receptor ligand sSpitz, is located in striking intrusions of the nuclear membrane. Here we characterize these structures and identify them as ‘cytoplasmic capes’, which were previously identified in old ultrastructural studies and probably coincide with recently recognized sites of non-nuclear-pore RNA export. We show that cytoplasmic capes contain multiple endosomal markers and that their existence is dependent upon βH and Annexin B9. Diminution of these structures does not lead to a change in mSpitz processing. These results extend the endosomal influence of βH and its partner Annexin B9 to this unusual compartment at the nuclear envelope

Semantic memory in Alzheimer's Disease

Alzheimer's Disease is characterized by a general decline in cognitive functioning. Although phonology are relatively unaffected, patients with Alzheimer's Disease have been reported to have deficits of semantic memory. Thirteen patients with dementia, five of whom had a confirmed diagnosis of dementia, participated in the study. The purpose of this investigation was to replicate a study performed by Mark Byrd (1984), using Alzheimer's Disease patients. Subjects were presented with category-word decision pairs, for which the task was to decide if the word was an exemplar of the category, and category-letter decision pairs for which the task was to generate an exemplar of the category beginning with the letter. The dependent variable was reaction time. Results indicated that Alzheimer's Disease patients and dementia patients had longer reaction times than a group of age-matched control subjects, and that the Alzheimer's Disease and dementia patients showed a pattern of responses similar to that of the control subjects. All groups showed longer reaction times for the generation trials than the decision trials. The results are consistent with the existence of a semantic memory deficit in Alzheimer's Disease, but other interpretations were discussed.Medicine, Faculty ofAudiology and Speech Sciences, School ofGraduat

Multiple Organ Failure After Ingestion of Pennyroyal Oil From Herbal Tea in Two Infants

Background Hepatic and neurologic injury developed in two infants after ingestion of mint tea. Examination of the mint plants, from which the teas were brewed, indicated that they contained the toxic agent pennyroyal oil. Methods. Sera from each infant were analyzed for the toxic constituents of pennyroyal oil, including pulegone and its metabolite menthofuran. Results. Fulminant liver failure with cerebral edema and necrosis developed in the first infant, who died. This infant was positive only for menthofuran (10 ng/mL). In the other infant, who was positive for both pulegone (25 ng/mL) and menthofuran (41 ng/mL), hepatic dysfunction and a severe epileptic encephalopathy developed. Conclusions. Pennyroyal oil is a highly toxic agent that may cause both hepatic and neurologic injury if ingested. A potential source of pennyroyal oil is certain mint teas mistakenly used as home remedies to treat minor ailments and colic in infants. Physicians should consider pennyroyal oil poisoning as a possible cause of hepatic and neurologic injury in infants, particularly if the infants may have been given home-brewed mint teas.</jats:p

Cytoplasmic capes contain ubiquitylated proteins and endosomal markers and are found in wild-type glands.

<p>Each set of three images shows one nucleus or cape from a salivary gland showing the distribution of two proteins. The center panel is a merged red/green image with the left panel in green. Due to the varied morphology of each cape multiple examples are shown for most markers. A–P express mSPI::GFP and costaining is for the indicated proteins: <b>A-A”</b> – Nucleoplasmic RFP is excluded from capes (arrowheads). This image is a single plane from a nucleus where the image stack had been deconvolved; <b>B-B”</b> – Wheat germ agglutinin (WGA) stains glycosylated proteins of the nuclear envelope and outlines the capes indicating that they are infoldings of this membrane and that the contents are therefore extranuclear. <b>C–F</b> – Lamin C similarly coats the cape membranes. D,E show individual capes from other nuclei. Panel F shows a series of confocal images taken at 0.5 μm intervals through a cape at the top of a nucleus. Chambers associated with individual capes where mSpitz is very low or absent are often seen with this marker (arrowheads). <b>G–H”</b> – Ubiquitin (Ubi) puncta are found in most capes at their periphery (see also P–Q”). H shows a saggital view. <b>I–J”</b> – Occasional Hrs puncta are found in the central space of the terminal chamber; <b>K–O</b> – EPS15 puncta are found in most capes in the central space. L–O show individual capes from other nuclei. Panels P–U” do not express mSpi::GFP demonstrating that capes are present in wild-type glands. Costainings are as indicated: <b>P–Q”</b> – Ubiquitin (Ubi) puncta surround Hrs puncta; <b>R–S”</b> – BicD::GFP is not obviously punctate and fills the central space containing EPS15 puncta. Note the large central vesicle that excludes both markers in example R. <b>T-T”</b> – Rab5 puncta are found in the central space; <b>U-U”</b> – Rab6 is not obviously punctate and fills the central space. Several other Rab proteins do not enter the capes. Due to the widely varying extent and morphology of each cape, some panels show single confocal planes whereas others are maximum projections of up to 8 planes taken at 1 μm intervals. All scale bars represent 10 μm except for panels A”, T’, T” and U” which are 20 μm.</p

A generalized drawing of a cytoplasmic cape.

<p>All wild-type capes have two regions: (1) A complex area of membrane folding at the nuclear periphery that contains abundant perinuclear vesicles. These vesicles are bound by a single membrane and separated from the nucleoplasm by a single membrane bilayer; (2) A terminal chamber where the two membrane bilayers separate single membrane bound organelles and granules (with no obvious membrane layer) from the nucleoplasm. The universal juxtaposition of these two regions seen by extensive serial sectioning leads us to hypothesize that their formation and perhaps function is linked. Blue line – Outer nuclear membrane; Red line – inner nuclear membrane.</p

Cytoplasmic capes are dependent upon β_H and AnxB9, but sSpitz production is not affected by β_H knockdown.

<p><b>A</b> – A wild-type salivary gland expressing mSpitz::GFP (AB1>mSpitz::GFP); <b>B,C</b> – Glands expressing mSpitz::GFP where β_H was knocked down (AB1>mSpitz::GFP+kst^RNAi). Just the occasional cape is seen at this resolution. <b>D</b> – Immunoblot on dissected salivary glands expressing mSpitz::GFP with or without β_H knockdown. Five pairs of glands are loaded for each genotype. In the upper panel the blot was probed with an anti-GFP antibody. mSpitz::GFP runs at about 58 kDa and the cleaved ligand, sSpi at 50kDa. The lowest band is non-specific. The lower panel is the same blot probed for Actin as a loading control. The amount of processed sSpi is not to be affected by the loss of the capes. <b>E</b> – The wing pouch region from a wild-type wing disc (MS1096>mSpitz::GFP). mSpitz::GFP marked nuclear envelopes have a rough, slightly punctate appearance. <b>F</b> – The wing pouch region from a wing disc where β_H was knocked down (MS1096>mSpitz::GFP+kst^RNAi). mSpitz::GFP now smoothly coats the nuclei. All images are maximum projections of multiple confocal sections. Scale bars represent 10 μm (E–F) or 20 μm (A–C).</p

Ultrastructural analysis of cytoplasmic capes.

<p>TEM images from three capes in wild-type nuclei. <b>A, B</b> – These images shows two capes where regions of perinuclear vesicles (PNV) and the terminal chamber (TC) are visible. NE – nuclear envelope. NP – nucleoplasm. As illustrated in A the PNV region is often associated with an out folding of the outer nuclear membrane (arrowhead), non-membrane bound granules (asterisk) and small vesicles (see D). Scale bars are 1 μm. <b>C</b> – Higher magnification view of part of the membrane from a terminal chamber in B (arrow) showing two membrane bilayers (arrowheads). Scale bar is 200 nm. <b>D</b>–A terminal chamber containing several vesicular and granular organelles (dashed line, asterisk). Two PNV are also visible at the periphery of this chamber (arrows). <b>E</b>–A second cape showing only the region of PNV. Scale bar is 2 μm. <b>F</b> – Higher magnification view of several PNV in C (dashed line with arrow). PNV are bounded by a single bilayer and are separated from the NP by a single bilayer and often protrude into one another. See also serial sections in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093680#pone.0093680.s003" target="_blank">Movie S1</a>. <b>G</b> – Chart showing the number of capes per nucleus with a given size (arbitrarily estimated in the Z dimension in serial block face SEM image series as the number of 50 nm sections from first appearance to disappearance in serial sections and normalized to the nuclear diameter in the same direction; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093680#pone.0093680.s004" target="_blank">Movie S2</a>). Data are shown for wild-type (black bars) and AnxB9^RNAi (grey bars). Many more small capes are present when AnxB9 is knocked down. <b>H</b> - Chart showing the number of fully sectioned nuclei containing capes with a terminal chamber. Data are shown for wild-type (black bars) and AnxB9^RNAi (grey bars). Whereas all capes end in a terminal chamber in wild-type, few do when AnxB9 is knocked down.</p