Video_3_Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization.AVI

<p>Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.</p

Video_1_Neuronal Growth Cone Size-Dependent and -Independent Parameters of Microtubule Polymerization.AVI

<p>Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.</p

MAP1B and syntrophin co-localize at the nodes of Ranvier and the abaxonal Schwann cell membrane.

<p>Sciatic nerves were prepared from 4-day (<i>4d</i>) or 14-day (<i>14d</i>) old or adult (<i>adult</i>) wild-type (<i>WT</i>) and MAP1B^−/− (<i>KO</i>) mice. Individual myelinated axons were isolated and stained for MAP1B (antibody anti-HC750) or syntrophin (pan syntrophin antibody anti-syn1351) as indicated. The pictures represent projections of confocal Z-stacks. The staining for MAP1B in postnatal and adult Schwann cells is specific as it is absent in Schwann cells of MAP1B^−/− mice. At all ages MAP1B was found to be concentrated at the nodes of Ranvier (<i>asterisks</i>). It also localized at the abaxonal membrane (<i>arrow heads</i>), particularly strong at postnatal day 14. Syntrophin was also found at nodes of Ranvier and the abaxonal membrane. In the adult, it was found to be localized to Cajal bands (<i>arrows</i>) in agreement with previous results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049722#pone.0049722-Albrecht1" target="_blank">[44]</a>. Co-localization of MAP1B and syntrophin was most prominent at the nodes of Ranvier and partial co-localization was found at the abaxonal membrane (<i>arrow heads</i>). Scale bar, 20 µm.</p

α1-syntrophin binds to microtubules in cells expressing LC1.

<p>PtK2 cells were transiently transfected to express either EGF-tagged α1-syntrophin alone (a and b) or α1-syntrophin and myc-tagged LC1 (c–e). Cells were fixed, co-stained for tubulin (<i>anti-tubulin</i>) and LC1 (<i>anti-myc</i>) and analyzed by fluorescence microscopy. In the absence of ectopically expressed LC1, α1-syntrophin was diffusely distributed throughout the cytoplasm (b). When co-expressed with LC1, α1-syntrophin was found to co-localize with LC1 on microtubules (c-e, arrows). Expression of LC1 causes microtubules to bundle, as has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049722#pone.0049722-Tgel1" target="_blank">[5]</a>. Scale bar, 20 µm.</p

α1-syntrophin is found in a complex with LC1 in the central and peripheral nervous system.

<p>Brain protein extracts obtained from transgenic mice expressing myc-tagged LC1 were immunoprecipitated (<i>IP</i>) either with anti-myc antibodies (<i>anti-myc</i>) or without antibody (<i>no ab</i>; negative control). Pellets (<i>P</i>) and the corresponding supernatants (<i>S</i>) were fractionated by SDS-PAGE and analyzed by immunoblotting (<i>WB</i>) using anti-syntrophin (<i>anti-syn1351</i>) or anti-myc antibodies (<i>anti-myc</i>). The positions of protein size markers, syntrophin, and LC1 are indicated. The double band corresponding to LC1 resulted from insufficient denaturation prior to gel electrophoresis.</p

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) 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

(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

Additional file 1 of Plectin plays a role in the migration and volume regulation of astrocytes: a potential biomarker of glioblastoma

Additional file 1: Table S1. Percentage of cells expressing astrocyte markers. Table S2. Primers used for RT-PCR