EMBO/soda-nel huggingface dataset:
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration (2020)
Figure 1

Figure 1 The BC network transiently expands during liver regeneration

A Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH. Shown are maximum projections of 50 µm z-stacks covering an entire CV-PV axis (CV, left; PV, right). Indicated regions of the CV (orange) and PV (blue) areas are shown as magnifications on the left and right, respectively.

B Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) in livers from untreated animals or mice at indicated timepoints post PH. The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=3-6 mice per timepoint. BC diameter of untreated mice vs. 0.8 d, p=1.62*10-11; untreated mice vs. 1.5 d, p=1.18*10-12; untreated mice vs. 2.0 d, p=5.69*10-11; untreated mice vs. 3.0 d, p=6.92*10-12; untreated mice vs. 5.0 d, p=1.65*10-7.

C EM images of BC on liver tissue sections of untreated mice (left) and 1.8 d post sham OP (middle) or PH (right).

D, E Quantification of BC perimeter (d) and total BC membrane length (e) from EM images as representatively shown in (c). Box-whisker plot with median, 25-75 quartiles and minimum/maximum error bars, n=5-6 mice per condition. In (d), BC perimeter of untreated vs. sham condition, p=0.94 (n.s.); Untreated vs. PH condition, p=0.01. In (e), BC membrane length of untreated vs. sham condition, p=0.86 (n.s.); Untreated vs. PH condition, p=0.03.

F IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL. Shown are maximum projections of 50 µm z-stacks in the CV (left, orange) and PV (right, blue) region. Images in (a) and (f) are background-subtracted. Scale bars, 50 µm (a), 1 µm (c), 5 µm (f).
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration (2020)
Figure 2

Figure 2 BC network dilation is accompanied by increased apical myosin levels during regeneration

A, C Fluorescence stainings for F-actin (a) or pMLC (c) and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm

B, D Quantification of apical F-actin (b) and pMLC (d) intensity from images as representatively shown in (a,c) as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 (b) and n=2-5 (d) mice per timepoint. Apical F-actin of sham vs. PH time course, p=0.03; Apical pMLC of sham vs. PH time course, p=0.003.
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration (2020)
Figure 3

Figure 3 BC network expansion coincides with activation of YAP during regeneration

A, B Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Indicated regions (orange squares) are shown as magnifications in (b). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.

C Quantification of the mean nuclear YAP (green) and PCNA (magenta) intensity from images of liver tissue sections at indicated timepoints post PH (solid line) and sham OP (dashed line) as representatively shown in (a) and Appendix Fig. S4A. Data was normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of PH vs. sham time course, p=2.97*10-5. Nuclear PCNA intensity of PH vs. sham time course, p=7.51*10-6.

D Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in (a) and Appendix Fig. S4A, at indicated time points post PH (solid line) or sham OP (dashed line) in 10 zones within the CV-PV axis (zone 1, CV area; zone 10, PV area). Data is normalized to untreated animals (not shown). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of sham vs. PH mice at 0.8d, p=5.35*10-5; 1.5d, p=3.95*10-5; 2d, p=5.99*10-5; 3d, p=2.16*10-5; 5d, p=0.01.

E Fluorescence stainings for YAP, CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH. Arrows indicate BC. Note the enrichment of apical YAP at 0.5 and 1.5d post PH.

F Fractionation of liver tissue at 2d post PH or sham (Sh) OP and untreated (UT) mice for nuclear (Nuc), cytoplasmic (Cyto) and membrane (Mem) fraction. TRF2, transferrin receptor2 (membrane marker); Histone H3 (nuclear marker); GAPDH, Glyceraldehyde 3-phosphate dehydrogenase (cytoplasmic marker).

G Quantification of YAP in the nuclear, cytoplasmic and membrane fraction from immunoblots as representatively shown in (f). YAP signal is normalized to the fraction marker (GAPDH, cytoplasm; Histon H3, nuclei; TRF2, membrane) and to the untreated condition (not shown). n = 3 (sham) or 4 (PH); data is shown as mean +/- s.e.m. Differences between sham and PH conditions for cytoplasmic, nuclear and membrane fractions are not significant.

Images in A, B and E are background-subtracted. Scale bars, 50 µm (A), 5 µm (B,E).
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration (2020)
Figure 4

Figure 4 YAP enriches in F-actin rich areas in the apical compartment of hepatocytes during regeneration

A EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles.

B, C Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.

The image in (B) is background-subtracted. Scale bar, 0.5 µm (a), 0.1 µm (a', a''), 2 µm (c), 0.2 µm (c'). BC, bile canaliculus; S, sinusoid.
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration (2020)
Figure 5

Figure 5 Bile acid induced YAP activation is dependent on the acto-myosin system

A Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets.

B Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05

C Western blot of pMLC and GAPDH (loading control) of primary hepatocyte culture lysates. Cells were untreated or incubated for 18 h with the indicated compounds

D Fluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment.

E Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92.

F Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration (2020)
Figure 6

Figure 6 Inhibition of Rho kinase reduces YAP nuclear accumulation during regeneration

A Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH, treated with saline (control) or the Rho kinase inhibitor Fasudil for 1 h. Shown are maximum projections of 50 µm stacks in the PV area.

B Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) from mice at 2 d post sham OP (green) or PH (red), treated with saline (control, solid line) or Fasudil (dashed line). Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. BC diameter of sham operated mice treated with saline vs. Fasudil, p= 6.59*10-8; BC diameter of PH mice treated with saline vs. Fasudil, p=2.31*10-14.

C Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH, treated with saline (control) or Fasudil for 1 h. Indicated regions (dashed rectangle) are shown as magnifications in insets.

D Quantification of the mean nuclear YAP intensity within 10 zones along the CV-PV axis, (zone 1, peri-central; zone 10, peri-portal) from IF images as representatively shown in (c). Data was normalized to untreated animals. Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. Nuclear YAP intensity of sham operated mice treated with saline vs. Fasudil, p=0.13 (n.s.) ; nuclear YAP intensity of PH mice treated with saline vs. Fasudil, p=0.0004.

Scale bars, 5 µm (A), 50 µm (C).
Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. (2013)
Figure 1

Chloroquine (CQ) treatment blocks autophagosome–lysosome fusion and induces myopathy in the larva.

(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green). (B–D) GFP–Atg8, overexpressed using the Dmef2–Gal4 driver, labels autophagosomes. Dmef2–Gal4, UAS–GFP–Atg8 animals were fed on high-nutrient food (B), starved on low-nutrient food for 6 h (C), or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (D). GFP–Atg8-labeled vesicles appeared only in the starved animals (C–D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP–Atg8-labeled vesicles. (E–G) Dmef2–Gal4, UAS–GFP–Atg8/UAS–HRP–Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP–Atg8 and HRP–Lamp1-labeled vesicles. (F) Colocalization of GFP–Atg8 and HRP–Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP–Atg8 and HRP–Lamp-labeled vesicles, but they failed to colocalize. (H) Quantification of the number of GFP–Atg8, HRP–Lamp1, or GFP–Atg8+HRP–Lamp1 vesicles in starved or starved +CQ muscles. (I–M) The core Atg genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. Dmef2–Gal4, UAS–GFP–Atg8/UAS–Atg1 larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that Atg1 knockdown completely abolished the formation of GFP–Atg8-labeled autophagosomes (compare I–J to C–D). (K–L) Dmef2–Gal4, UAS–GFP–Atg8, Atg1Δ3d larvae failed to form GFP–Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to Atg gene knockdown in Dmef2–Gal4, UAS–GFP–Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 UAS–Atg RNAi transgenes tested caused a highly significant decrease (p<.01) in the total area of GFP–Atg8 vesicles. SEM is indicated, with n = 5 ventral longitudinal muscles from individual animals. (N–O) EM of muscles from Dmef2–Gal4, UAS–whitei larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N). (P) CQ treatment increased the larval crawling time of Dmef2–Gal4, UAS–whitei larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of Dmef2–Gal4, UAS–whitei larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for n = 10 larvae (*p<.05, **p<.01).
Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. (2013)
Figure 2

Autophagosomes in the larval muscle are filled with glycogen.

(A) Sectioned third instar OreR larva stained with Periodic acid-Schiff (PAS). The muscles, but not the fat body, are stained purple, indicating high levels of glycogen (m, muscle; bw, body wall; fb, fat body). (B) Glycogen was also detected in muscle from Dmef2–Gal4, UAS–GFP–Atg8 larvae, immunostained with an antiglycogen monoclonal antibody. (C) GFP–Atg8 vesicles colocalized with glycogen in Dmef2–Gal4, UAS–GFP–Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h (GFP, green; antiglycogen, red). (D) HRP–Lamp1 vesicles show less colocalization with glycogen in UAS–HRP–Lamp1;Dmef2–Gal4 larvae starved and treated with CQ. (E) Quantification of GFP–Atg8 or HRP–Lamp1 vesicles with glycogen. (F–G) EM from Dmef2–Gal4, UAS–whitei larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2–Gal4, UAS–whitei larva starved on low-nutrient food for 6 h. Arrow points to double membrane.
Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. (2013)
Figure 3

Degradation of glycogen via the autophagy lysosome system is regulated by nutrients and the Tor pathway.

(A–D) Time course of autophagy induction in Dmef2–Gal4, UAS–GFP–Atg8 muscles, accompanied by quantification of GFP–Atg8 and glycogen colocalization. Animals were fed for 18 h in high-nutrient food +2.5 mg/ml CQ, then starved on low-nutrient food +2.5 mg/ml CQ for 0–8 h (antiglycogen, red; GFP, green; DAPI, blue). (A) At time point 0, following 18 h in high-nutrient food +CQ, the muscles contained large amounts of glycogen with no apparent autophagy. (B) At 3 h of starvation, glycogen stores were still high, and GFP–Atg8-labeled vesicles began to appear. (C–D) At 6 and 8 h of starvation, the majority of GFP–Atg8-labeled vesicles colocalized with glycogen. (E) Time course of glycogen levels in Dmef2–Gal4 carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0–24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for n = 5–8 samples (*p<.05, **p<.01). (F–G) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Autophagy levels were high in control Dmef2–Gal4/UAS–whitei larvae. Muscles from (G) UAS-Rheb/+; Dmef2-Gal4/+, (H) Dmef2–Gal4/UAS–Tsc1i, and (I) Dmef2–Gal4/UAS–gigi all failed to induce autophagy.
Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. (2013)
Figure 4

Autophagy and glycogenolysis compensate for each other, but both systems are required for maximal glycogen catabolism.

(A–B) Glycogen phosphorylase is not required for glycogen autophagy (antiglycogen, red; GFP, green; DAPI, blue). (A) UAS–GlyPi/+; Dmef2–Gal4, UAS–GFP–Atg8/+ larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h exhibited high levels of colocalization between GFP–Atg8 and glycogen. (B) Higher magnification of region outlined in (A). (C–F) Dmef2–Gal4, UAS–GFP–Atg8 larvae with GlyP and/or Atg1 knockdown were fed on high-nutrient food for 18 h before being starved on low-nutrient food (antiglycogen, red; GFP, green; DAPI, blue). (C) UAS–GlyPi/+; Dmef2–Gal4, UAS–GFP–Atg8/+ larval muscle contained high levels of glycogen prior to starvation, indicating no defect in glycogen synthesis. (D) Following 24 h starvation UAS–GlyPi/+; Dmef2–Gal4, UAS–GFP–Atg8/+ muscles contained no glycogen detectable by antibody staining. (E) Following 24 h of starvation Dmef2–Gal4, UAS–GFP–Atg8/UAS–Atg1i muscles contained no glycogen. (F) Double-mutant larvae UAS–GlyPi/+; Dmef2–Gal4, UAS–GFP–Atg8/UAS–Atg1i larval muscles contained high levels of glycogen after 24 h of starvation, indicating an inability to break down glycogen. (G) Time course of glycogen levels in Dmef2–Gal4 carcasses (muscle+body wall) with expression of UAS–RNAi transgenes targeting white, GlyP, Atg1, or GlyP+Atg1. Simultaneous knockdown of GlyP and Atg1, but not either gene alone, significantly reduced glycogen degradation compared to the white control after 24 h of starvation, consistent with immunostaining (C–F). Between 6 and 12 h of starvation, individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels, indicating a reduced rate of glycogen degradation. SEM is indicated for n = 5–8 samples. The p values were calculated relative to white RNAi control at each time point (*p<.05, **p<.01).
Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. (2013)
Figure 5

Glycogen synthase knockdown inhibits autophagosome growth and improves CQ-induced myopathy phenotype.

(A–D) Glycogen synthase (GlyS) is required for glycogen synthesis in D. melanogaster muscles. PAS staining for glycogen was absent in Dmef2–Gal4/UAS–GlySi muscles (B) compared to control Dmef2–Gal4/UAS–whitei muscles (A). Antiglycogen immunostaining for glycogen was absent in Dmef2–Gal4/UAS–GlySi muscles (D) compared to Dmef2–Gal4/UAS–whitei control muscles (C). (E–K) GlyS is required for the formation of large CQ-induced autophagosomes. Vesicles are much smaller in Dmef2–Gal4, UAS–GFP–Atg8/UAS–GlySi larval muscle starved 6 h in low-nutrient food +2.5 mg/ml CQ (G) than in control Dmef2–Gal4, UAS–GFP–Atg8/UAS–whitei larval muscle (E). (F, H) The difference in autophagosome size is clearly evident at high magnification. (I–K) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2–Gal4, UAS–GFP–Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. SEM is indicated, with n = 5 (I) or n = 10 (J–K) ventral longitudinal muscles from individual animals (*p<.05, **p<.01). (I) Each of the four UAS-GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP–Atg8 vesicles in the muscle compared to the UAS–whitei control. (J) Vesicle number was unchanged by GlyS knockdown. (K) UAS–GlyS RNAi caused a highly significant decrease in the mean vesicle size (area) compared to the control. (L–M) GlyS gene expression, monitored using a MiMIC transposon insertion (MI01490), showed expression in the larval muscle (L) but not the fat body (M) (green, GFP; blue, DAPI). (N–O) Larvae were starved on low-nutrient food for 6 h prior to dissection of the fat bodies. Autophagy in Cg-Gal4/+; UAS–GFP–Atg8/UAS–GlySi (O) was not substantially different from autophagy in Cg-Gal4/+; UAS–GFP–Atg8/UAS–whitei control fat bodies (GFP, green; DAPI, blue). (P) EM of muscle from Dmef2–Gal4/UAS–GlySi animal starved on low-nutrient food +CQ. Note that the intermyofibril spaces (red asterisk) and sarcomere structure are not distorted. (Q) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h. SEM is indicated for n = 10 larvae. The p values were calculated relative to white RNAi control larvae (*p<.05, **p<.01).
Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. (2013)
Figure 6

Interaction and colocalization of Glycogen synthase with Atg8 is disrupted in R593A and W609A mutants.

(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined. (B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag–Atg8 binds to a Venus–GlyS or Venus–GlyS (S651A) protein complex in response to starvation. Flag–Atg8 is unable to co-IP with either Venus–GlyS (R593A) or Venus–GlyS (W609A). Venus–GlyS and Venus–GlyS mutants were co-IP'd from muscle lysate from Dmef2–Gal4/UAS–Flag–Atg8 or UAS–Venus–GlyS(WT or mutant)/+;Dmef2–Gal4/UAS–Flag–Atg8 third instar larvae. These were fed on high-nutrient food for 18 h, and then transferred to fresh high-nutrient food or low-nutrient food for 6 h. (C–F) UAS–Venus–GlyS (WT or mutant)/+;Dmef2–Gal4/UAS–Flag–Atg8 larvae were treated with starved 6 h in low-nutrient food +2.5 mg/ml CQ (Venus, green; α-Flag, red). Purple arrows mark examples of the presence or absence of colocalization. (C) Venus–GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus–GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus–GlyS (S651A) was localized to the autophagosomes. (F) Venus–GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.
Cryo-EM reveals the complex architecture of dynactin's shoulder region and pointed end (2021)
Figure 6

Figure 6: Pointed end cargo adaptor interaction sites

A. Site 1 in the disordered loop of p62 (orange, in clear density), binds to each cargo adaptor (BICDR1 in yellow, Hook3 in purple, BICD2 in green). Density can be seen between p62 and the adaptor at low threshold. BICDR1- and Hook3-containing maps are filtered to 6 Å.

B. Residues on dynactin's pointed end (cartoon) that interact with cargo adaptors are shown as spheres, colored according to which adaptor they bind (yellow denotes binding to BICDR1, purple to Hook3 and green to BICD2). Binding sites 2-4 are shown in boxes.

C. Conservation of the surface residues plotted onto the density from the pointed end map, filtered to 6 Å. Sites 2-4 for cargo adaptor interaction are marked on the front face and end face. Conservation scores are from ConSurf, with lower conservation shown in green, and higher conservation in purple.

D,E. Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.