Journal: Cells

Article Title: Sec20 Is Required for Autophagic and Endocytic Degradation Independent of Golgi-ER Retrograde Transport

doi: 10.3390/cells8080768

Figure Lengend Snippet: Sec20 is responsible for proper endocytic transport in garland nephrocytes. ( A , B ) Garland nephrocytes were co-stained against early endosome-specific Rbsn-5 and late endosome-specific Rab7. Late endosomes show significant enlargement upon Sec20 depletion ( A ) compared to control cells ( B ). ( C , D ) When nephrocytes were incubated with TexasRed-Avidin (TRA) fluorescent endocytic tracer for a 5 min pulse (5’p, without subsequent chase), the tracer appeared closer to the cell membrane in Sec20 RNAi cells ( D ) than in controls ( C ). ( E , F ) However, when cells were chased for 30 min following the 5 min pulse, TRA was transported to the perinuclear region both in control ( E ) or Sec20 knockdown ( F ) cells. ( G ) Quantification of the areas of Rab7-positive vesicles of the nephrocytes of each Sec20 RNAi lines compared to the same control case (note that the confocal images of Sec 20 RNAi2, RNAi3 RNAi nephrocytes are shown in ). ( H ) Quantification of the TRA uptake and transport assays from ( C – F ). ( G , H ). Medians are shown as horizontal black lines within the boxes. Bars show the upper and lower quartiles, and the whiskers plot the smallest and largest observations. ( I , J ) Garland nephrocytes were incubated with DQ-BSA during a 5 min pulse and were then chased for another 30 min. In controls, numerous red puncta indicate efficient proteolysis inside endolysosomes ( I ). Significantly fewer puncta were observed in Sec20-depleted cells ( J ). ( K , L ) Vps41-9xHA was overexpressed using pros-Gal4 in either control ( K ) or Sec20 RNAi expressing cells ( L ) and stained against Rab7 and HA. The colocalization between Vps41-9xHA with a subset of Rab7 positive endosomes is evident in controls ( K ). Vps41-9xHA sustains its late endosomal localization in cells undergoing Sec20 RNAi. NS: non-significant ( p ≥ 0.05), *** p < 0.001. Scale bars: 20 µm.

Article Snippet: For TexasRed-Avidin and DQ Red BSA uptake assays, L3 larval proventriculi with garland nephrocytes were prepared in cold Shields and Sang M3 insect medium (Merck, Darmstadt, Germany, S8398) and incubated in 0.1 mg/mL TexasRed-Avidin D (Vector Laboratories, A-2006) or 10 µg/mL DQ Red BSA (Thermo Fisher Scientific, Waltham, MA, USA, D12051) containing M3 for 5 min at RT, rinsed 3 times, and incubated in M3 for 30 min where stated, then fixed with 4% formaldehyde in PBS (50 min at RT).

Techniques: Staining, Incubation, Avidin-Biotin Assay, Expressing