Table_2_Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.XLSX

<p>Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.</p

Image_2_Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.TIF

<p>Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.</p

Table_1_Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.XLSX

<p>Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.</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) 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) 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

DS_10.1369_0022155418786698 – Supplemental material for Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution

<p>Supplemental material, DS_10.1369_0022155418786698 for Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution by Siegfried Reipert, Helmuth Goldammer, Christine Richardson, Martin W. Goldberg, Timothy J. Hawkins, Elena Hollergschwandtner, Walter A. Kaufmann, Sebastian Antreich and York-Dieter Stierhof in Journal of Histochemistry & Cytochemistry</p