Evaluasi Peran Sistem Pengendalian Manajemen untuk Meminimalkan Konflik pada Badan USAha Keluarga “K” di Tulungagung

Penelitian ini bertujuan untuk mengevaluasi peran sistem pengendalian manajemen untuk meminimalkan konflik. Penelitian ini merupakan applied research yang dilakukan menggunakan pendekatan kualitatif. Objek penelitian dalam penelitian ini yaitu badan USAha keluarga “K” di Tulungagung. Sumber data untuk penelitian ini adalah narasumber yang terdiri dari direktur, supervisor dan karyawan. Metode pengumpulan data yang digunakan adalah wawancara semi structured, observasi, dan analisis dokumen. Hasil evaluasi yang dilakukan dalam penelitian ini menunjukkan bahwa sistem pengendalian manajemen yang diterapkan oleh badan USAha “K” sudah berhasil meminimalkan konflik seperti substantive conflict. Namun ada beberapa bentuk pengendalian manajemen yang masih memiliki kelemahan yang berpotensi dan telah menimbulkan process conflict dan affective conflict khususnya di badan USAha “K” saat ini

p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions

<div><p>α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson’s disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated <em>in vitro</em>. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.</p> </div

The reduced incorporation of α-synuclein inclusions into autophagosomes by autophagy inhibition.

<p>(<b>A</b>) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm. (<b>B</b>) The number of GFP-LC3-positive α-synuclein inclusions (%) were assessed in randomly chosen fields (n = 5) with 48–141 inclusions. Statistical analysis was performed with <i>t</i>-test (*<i>p</i><0.01). (<b>C</b>) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *<i>p</i><0.01.</p

Autophagic clearance of α-synuclein inclusions.

<p>(<b>A</b>) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes. (<b>B</b>) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.</p

Impairment of mitochondria and mitophagy in cells harboring α-synuclein inclusions.

<p>(<b>A</b>) α-Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed-LC3 and EGFP-parkin. After 24 h, the cells were immunostained with anti-phosphorylated α-synuclein (P-αSyn). There was no alteration in the localization of EGFP-parkin. The arrows indicate α-synuclein inclusion. Scale bar, 10 µm. (<b>B</b>) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052868#pone.0052868.s008" target="_blank">Fig. S8</a>). Scale bar, 10 µm.</p

Involvement of p62 in the selective clearance of α-synuclein inclusions.

<p>(<b>A</b>) HEK293 cells stably expressing GFP-LC3 were transfected with p62-siRNA (right panels) or control-siRNA (left panels). Phosphorylated α-synuclein-positive inclusions (P-αSyn) are indicated by arrowheads. In p62-knockdown cells, the expression and accumulation of p62 were not detected, and inclusions were never sequestrated by GFP-LC3 autophagosomes. Scale bars, 20 µm. (<b>B</b>) The number of GFP-LC3-positive α-synuclein inclusions (%) in Fig. 5A were assessed in randomly chosen fields with 32–44 inclusions (n = 3). Data represent mean ± s.e.m. Statistical analysis was performed with <i>t</i>-test (*<i>p</i><0.05). (<b>C</b>) α-Synuclein fibrils were introduced into control (Cont) or p62-knockdown (p62) cells. Four hours after introduction, cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *<i>p</i><0.01. This experiment was repeated three times. (<b>D</b>) Clearance of α-synuclein inclusions in control (solid bar) or p62 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *<i>p</i><0.01.</p

Autophagic clearance of α-synuclein inclusions.

<p>(<b>A</b>) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *<i>p</i><0.05. This experiment was repeated three times. (<b>B</b>) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction. (<b>C</b>) The number of phosphorylated α-synuclein-positive cells per unit area (0.6 mm²) was assessed in randomly chosen fields (n = 6). Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *<i>p</i><0.05, **<i>p</i><0.001. (<b>D</b>) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm. (<b>E</b>) Colocalization of α-synuclein inclusions (n = 41–73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey’s test). *<i>p</i><0.01.</p

Formation of Lewy body-like α-synuclein inclusions in cultured cells.

<p>(<b>A</b>) Purified His-α-synuclein protein was agitated for 0–2 weeks (lane 1, 0weeks; lanes 2 and 3, 1week; lanes 4 and 5, 2weeks). Aliquots from each sample were separated into supernatant (lanes 2 and 4) and pellet (lanes3 and 5) fractions. These fractions were subjected to immunoblotting with anti-α-synuclein antibody. Numbers at left are molecular weight markers (MW) in kilodaltons. (<b>B</b>) Fibril formation of α-synuclein was measured with ProteoStat, an aggregation detection dye. a.u., arbitrary units. (<b>C</b>) AFM images of α-synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode. A high-magnification view of the boxed area is shown in the lower panel. The scale on the right represents the height of pixels in the image. Scale bars, 100 nm. (<b>D</b>) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.</p