Plectin 1d, 1f, 1b, and 1 link desmin IFs with Z-disks, costameres (DGC), mitochondria, and the outer nuclear/ER membrane system, respectively

<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Soleus f-ple (a and c) and cKO-ple (b and d) sections double immunolabeled for plectin and desmin (a and b) or stained for desmin alone (c and d)

Note, desmin aggregates in the fiber interior (d, arrow) and accumulates along the sarcolemma (d, arrowhead) in plectin-negative fibers. The double-headed arrow in panel b represents a plectin-positive fiber with a preserved desmin-positive pattern. (B) f-ple (a, c, and e) and cKO-ple (b, d, and f) heart sections immunolabeled using antibodies to proteins as indicated. In cKO-ple cardiomyocytes, note the aggregates of desmin (b, arrow) and misaligned Z-disks (f, inset) as well as the seemingly preserved intercalated disk structures (double arrows). (C) f-ple (a and c) and cKO-ple (b and d) soleus longitudinal (a and b) and EDL cross sections (c and d) stained for proteins as indicated. Asterisks indicate fibers devoid of IFs in the fiber interior. The double-headed arrow in panel b represents a CNF with preserved IF pattern. The dotted boxes in panels c and d indicate areas shown magnified in the insets. (D) Immunofluorescence microscopy of teased fibers from f-ple (a and c) and cKO-ple (b and d) EDL revealing massive longitudinal desmin aggregates (b) and misaligned α-actinin–positive costameres (d, inset) in cKO-ple mice. No misalignments were observed in the case of f-ple costameres (c, inset). Note also the close association of desmin IFs with f-ple nuclei (a, inset) but their detachment from cKO-ple nuclei (b, inset). Dotted boxes indicate areas shown magnified in insets. Bars, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Longitudinal sections of soleus immunostained using antiserum 46 to plectin

Striated plectin patterns are observed in ple1, ple1b, and dessamples; in ple1d and ple1d/des samples, such patterns are missing. The arrow and arrowheads in the ple1d panel represent plectin-positive sarcolemmal and interior dotlike structures, respectively. Note that the interior of ple1d/des fibers is completely devoid of plectin-positive signals. (B) Teased fibers of EDL were immunostained as in A. Note, the signal associated with longitudinal perinuclear structures was decreased in ple1 compared with ple1b fibers (arrows). Also, costameres were focally disorganized in ple1d and des samples (arrowheads). (C) Ple1d soleus sections double immunolabeled for plectin and desmin (a), desmin and mitochondria (b), or stained for SDH (c). Inset shows subsarcolemmal aggregation of mitochondria in a magnified view of the boxed area. The electron micrograph in panel d shows internal lysis of enlarged mitochondria in the subsarcolemmal region (arrows). (D) Ple1d EDL cross section double immunolabeled for desmin and synemin revealing aggregates in the interior of fibers and largely unaffected sarcolemmal regions (see also inset, a magnified view of the boxed area). (E) Immunofluorescence microscopy of teased ple1d fibers (EDL) using antibodies as indicated. In panels a and b, note the largely unaffected perinuclear and costameric patterns of plectin 1 and 1f, respectively. Panels c and c′ represent sequential confocal sections of one fiber. An optical cross section of this fiber (marked 1) is shown as an inset in panel c′, with horizontal lines indicating the positions of the planes shown in panels c and c′. Note the costameric patterns lacking aggregates in panel c and that desmin aggregates in the interior part of the fiber in panel c′ (arrow). Bars: (A; B; C, a and b; D; and E) 20 μm; (C, c) 50 μm; (C, d) 2 μm. (F) Quantitative immunoblotting of plectin in gastrocnemius lysates from different mouse mutants. Data, relative to WT samples (100%), represent the means ± SEM of three experiments.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Representative regions of teased EDL fibers from 4-mo-old f-ple and cKO-ple mice stained for proteins as indicated

Arrowheads and arrows indicate Z-disk–aligned and perpendicular longitudinal desmin-positive costameric structures, respectively. In f-ple fibers, note the colocalization of desmin IFs with syncoilin, synemin, cytokeratin 8, β-DG, dystrophin, nNOS, and syntrophin but not with caveolin 3. In cKO-ple fibers, all costameric marker proteins show profoundly changed localization patterns. Bar, 5 μm. (B and C) Quantitative immunoblotting analysis of gastrocnemius lysates from three 6-mo-old mice per genotype (B) and of microsomal fractions from at least three gel runs (C). Loading was normalized to total protein contents (Coomassie-stained gels). Bar graphs represent mean values ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

MCT8 and D3 immunoreactivities in axon varicosities of the rat parvocellular hypophysiotropic neurons.

<p>Confocal images were subjected to deconvolution. Boxed areas are enlarged in <b>B</b> and <b>C</b>, respectively. The immunofluorescent signal for MCT8 (red) is distributed as small dots throughout the external zone of median eminence and appears (arrowheads) on the surface of gonadotropin-releasing hormone (GnRH, <b>C</b>), thyrotropin-releasing hormone (TRH, <b>D</b>), corticotropin-releasing hormone (<b>E</b>), growth hormone-releasing hormone (GHRH, <b>F</b>) and somatostatin (SST, E) immunofluorescent axon varicosities (green). (<b>H,I</b>) Representative images of triple immunofluorescent labelings demonstrate D3 (blue), MCT8 (red) and a hypophyseotroph hormone (green) in the axons of the median eminence. MCT8-immunoreactive puncta (arrowheads) appear on the surface of the following categories of axon varicosities; single-labeled for D3 (<b>Ha</b>), single-labeled for GnRH (<b>Hb</b>), and double labeled for D3 and GnRH (<b>Hc</b>) or CRH (<b>I</b>). Scale bars: A: 10 µm, B: 5 µm, C-I: 2 µm.</p

Schematic illustration of axonal uptake and regulation of T3 in the mediobasal hypothalamus.

<p>We suggest that T3 generated by D2 is released from tanycyte processes and taken up by MCT8 into axons of hypophysiotropic neurons. T3 concentrations are subjected to local regulation by D3-containing axon varicosities (At1), but absent in D3-negative axons (At2). T3 could be subjected to retrograde transport to reach the soma and nucleus of hypophysiotropic neurons and/or could act locally by affecting mitochondrial function and local thermogenesis. At, axon terminal; Tc, tanycyte; Pc, portal capillary.</p

Ultrastructure of D3 immunoreactive elements in the rat mediobasal hypothalamus.

<p>(<b>A</b>) D3 immunoreactivity, identified by silver grain deposits, appear primarily in axon varicosities containing dense core vesicles of 80–120 nm diameter in the upper external zone of the median eminence, characteristic of axons of parvocellular (PC) neurons. No or a few silver grains could be observed in association with organelles of magnocellular neurons (MC) or tanycytes (Tc), respectively. (<b>B</b>) D3-positive axons exhibiting various degrees of labeling are mixed with non-labeled fibers (asterisks) in the external zone of the median eminence. (<b>C</b>) Although silver grains occasionally appear in association with the plasma membrane (black arrowheads) and with small clear vesicles (white arrowheads) of the axon varicosities, the majority are not labeled (asterisks). (<b>D</b>) In contrast, the dense core vesicles accumulate most the reaction product (black arrowheads), as visible at high power magnification in the vicinity of the capillaries of the external zone of the median eminence. Unlabeled small clear vesicles are indicated with asterisk. Tc, tanycyte; Scale bars: 1 µm in A–B, 250 nm in C, 100 nm in inset on C, 500 nm in D.</p

D3 expression in processes of mouse immortalized GT 1-7 GnRH neurons <i>in vitro.</i>

<p>(<b>A</b>) GT 1-7 cells endogenously express D3 mRNA. –RT: minus reverse transcriptase control; + and -: positive and negative controls, respectively (<b>B</b>) Schematic illustration of Bimolecular Fluorescence Complementation used to study D3 homodimerization. YFP(N) and YFP(C) stand for YFP(1-158aa) and YFP(159–238aa), respectively. (<b>C</b>) GT 1-7 cells were transfected with a plasmid encoding the fusion protein D3-YFP(full-length). (<b>D</b>) Co-expression of YFP(1-158)-D3 and YFP-(159–238)-D3 fusion proteins results in fluorescence complementation and demonstrates D3 homodimers in the axon-like processes of GT1-7 cells. Arrows indicate D3 along the processes. Scale bars: 10 uM. (<b>E</b>). Axonal D3 activity is increased by T3-treatment in the median eminence of male Wistar rats. Mean±SEM (n = 9) *P<0.05 by t-test.</p

D3-immunoreactivity in the infundibular stalk of the human hypothalamus.

<p>(<b>A</b>) D3 immunoreactive fibers (arrowheads) are present in the human infundibular stalk (IS); these fibers are shown in medium (<b>B</b>) and high (inset in A) power micrographs. opt: tractus opticus, VMH: ventromedial hypothalamic nucleus, III: third ventricle Scale bars: 500 µm in A, 50 µm in B, 10 µm in inset.</p

Dual-immunofluorescence images illustrate the overlapping distribution of fibers immunoreactive for D3 (green fluorochrome) and GnRH (A) or TRH (D) (red color) in the median eminence.

<p>Sites of overlap (yellow color) occur along the axonal pathway in the median eminence (<b>B,E</b>). High power confocal images demonstrate dual-labeled axon varicosities (yellow color in composite images) immunoreactive for D3 and GnRH (<b>C</b>) or D3 and TRH (<b>F</b>). The D3 immunoreactivity appears as yellow patches (arrowheads) within the axon varicosities. Single channels are also shown in <b>C’, C”</b> and <b>F’, F”</b>, respectively. High power dual-immunofluorescent images are also shown for fibers labeled for D3 (green fluorochrome) and CRH (<b>G</b>), GHRH (<b>H</b>) or somatostatin (<b>I</b>) (red color). These D3-immunoreactive sites (arrowheads) correspond to axon varicosities (<b>G</b>, <b>H</b>). Somatostatin (SS)- immunoreactive axon varicosities show virtually no signal for D3. Scale bars: 20 µm in A, 5 µm in B, 5 µm in C, 10 µm in D,E, 5 µm in F–I.</p