Figure 1.
Kalirin-7 interacts with synphilin-1 in vitro and in vivo.
(A) Mapping of the interacting domain in the kalirin-7 protein. FLAG-kalirin-7 constructs as shown in the diagram were co-transfected with V5-synphilin-1 in HEK293 cells. 24 h after transfection, cells were subjected to immunoprecipitation with anti-FLAG agarose beads and subsequently kalirin-7 and synphilin-1 immunoreactivities were monitored applying anti-FLAG- or anti-V5 antibodies, respectively. IP indicates antibodies used for pulling down target proteins. IB indicates antibodies used for detection in western blot. The figure shows that kalirin-7 co-immunopreciptates with synphilin-1 and that spectrin repeats III and IV of the kalirin-7 protein are crucial for the interaction. Quantification of kalirin-7 fragment expression is shown in Figure S3. (B) Mapping of the binding region in synphilin-1. The indicated V5-synphilin-1 constructs were co-transfected with FLAG-kalirin-7. Synphilin-1 fragments were precipitated with anti-V5 antibodies. The precipitates were then probed with anti-FLAG antibodies to detect co-precipitated kalirin-7. The deletion mapping revealed that amino acids 1–348 of the synphilin-1 protein are crucial for the binding of kalirin-7. The asterisks indicate specific input signals of synphilin-1 fragments. For quantification of synphilin-1 fragment expression please refer to Figure S3. (C) Endogenous synphilin-1 interacts with kalirin-7. Synphilin-1 was precipitated from whole-brain tissues (500 µg) of a wild type mouse with an anti-synphilin-1 antibody (Sigma). The precipitates were probed with a kalirin-7-specific antibody (KALRN from Abcam). Cell lysate of HEK293 cells overexpressed with FLAG-kalirin-7 and V5-synphilin-1 served as positive control. As a negative control brain lysate was subjected to immunoprecipitation without antibody. (D) Overlapping localization of kalirin-7 and synphilin-1 in cell culture. HEK293 cells were transiently transfected with both constructs for 6 h and stained with anti-FLAG and anti-V5 antibodies. The counterstaining was done with YoPro dye. The confocal sections demonstrate that both proteins display a punctate staining in the cytoplasm. Sph1, synphilin-1; Kal7, kalirin-7. Scale bar, 10 µm.
Figure 2.
Kalirin-7 alters synphilin-1-induced inclusion formation.
(A) When HEK293 cells were transfected with HcRed-synphilin-1 alone (a,b), FLAG-kalirin-7 (c) or both expression constructs (d) for 48 h, two types of inclusions were observed: small cytoplasmic aggregates (arrowhead; a) and perinuclear aggregates (arrow; b, d). Blue, DAPI. Scale bar, 10 µm. (B) Quantitative analysis of the experiment described in (A). HcRed-synphilin-1 was expressed without or with FLAG-kalirin-7 for 48 h. Cells were fixed and immunostained with anti-FLAG antibodies. Cells with cytoplasmic small aggregates, perinuclear aggregates or soluble synphilin-1 were counted. Results represent the average of three independent experiments. (C) Total numbers of aggregates per cell (cytoplasmic and perinuclear) were counted applying ApoTome confocal fluorescent microscopy. Over 100 cells were counted for each condition. The asterisks indicate statistical significance (**P≤0.005; ***P≤0.001). Error bars, S.E.
Figure 3.
Kalirin-7 mediates perinuclear synphilin-1 inclusion formation in a microtubule-dependent manner.
(A) HEK293 cells were cotransfected with HcRed-synphilin-1 and FLAG-kalirin-7. After 36 h cells were incubated with DMSO, 5 µM nocodazole or 10 µM colchicine for 12 h before being subjected to immunofluorescence with anti-FLAG and anti- β-tubulin antibodies. Cells expressing HcRed-synphilin-1 alone served as controls (arrowhead). In cells treated with nocodazole or colchicine, more cytoplasmic small aggregates (arrows) were formed. (B) Quantification (n >250 cells per group) shows that nocodazole and colchicine inhibited the kalirin-7-mediated formation of synphilin-1-containing perinuclear inclusions. P, perinuclear aggregates; C, cytoplasmic small aggregates. The asterisks indicate statistical significance (**P≤0.005). Error bars, S.E.
Figure 4.
Characterization of synphilin-1-containing aggregates as aggresomes.
HEK293 cells coexpressing HcRed-synphilin-1 and FLAG-kalirin-7 were fixed 48 h post-transfection and subsequently stained with the indicated antibodies. Arrows indicate the colocalization between synphilin-1 inclusions and γ-tubulin, ubiquitin and Hsp27 while the intermediate filament protein vimentin forms a cage surrounding a pericentriolar core of aggregates. Merged images are shown to the right. Blue, DAPI. Scale bar, 10 µm.
Figure 5.
Kalirin-7 decreases synphilin-1-induced aggregates in biochemical and live cell analysis.
(A) HEK293 cells were transfected with HcRed-synphilin-1 alone or cotransfected with FLAG-kalirin-7. HcRed empty vector served as control. Cells were lysed 24, 48 or 72 h after transfection, fractionated by AGERA on 2% agarose gels and analyzed by western blotting with an antibody recognizing synphilin-1 aggregates. S, HcRed-synphilin-1; K, FLAG-kalirin-7; C, control (HcRed empty vector). Indicated by a bracket on the right is the the major area of aggregate signal which was used for quantification. (B) Quantification of AGERA blots of 3 independent experiments for each time point and condition relative to the mean expression level of controls at 24 hrs post-transfection confirmed an increase of aggregates over time and a reduced number of aggregates in cells doubly transfected with kalirin-7 and synphilin-1 compared to cells transfected with synphilin-1 alone at 48 and 72 hours. (C) Long-term time-lapse imaging. HEK293 cells were transfected with HcRed-synphilin-1 and empty EGFP vector (upper chart) or EGFP-kalirin-7 (lower chart) and observed by live cell imaging fluorescent microscopy (Cell Observer, equipped with an Axio Observer.Z1 and an ApoTome Imaging System Zeiss, Germany) at 37°C. Depicted are average intensity projections of 6–8 ApoTome optical slides encompassing the entire height of the cells. Time-points indicate hours post-transfection. Images were merged from red, green and phase contrast channels. Arrows indicate the cell traced over the experimental time. Scale bar, 10 µm. (D) Quantification (n >35 cells per group) of the time-lapse imaging shows that aggregate numbers are reduced when FLAG-kalirin-7 is coexpressed. Light gray bars: Sph alone; dark gray bars: Sph and Kal7 coexpression. Results represent the average of three independent experiments. The asterisks indicate statistical significance (*P≤0.05). Error bars, S.E.
Table 1.
Percent of cells with cytoplamic small aggregates or perinuclear aggregates in HEK293 cells transfected with different small GTPase competitive constructs.
Figure 6.
Kalirin-7-mediated recruitment of synphilin-1 inclusions into aggresome is blocked by the HDAC inhibitor trichostatin A.
(A) HEK293 cells expressing HcRed-synphilin-1 (a,b,c) or co-expressing HcRed-synphilin-1 and FLAG-kalirin-7 (d,e,f) were incubated in the presence of DMSO (a,d), 1 µM TSA (b,e) or 5 mM NaBu (c,f) for 18 h before being fixed and immunostained with anti-FLAG antibodies. The arrow indicates synphilin-1 cytoplasmic small aggregates. Blue, DAPI. Scale bar, 10 µm. (B) Quantification shows that treatment with the HDAC6 inhibitor TSA counteracts the recruitment of synphilin-1 into aggresomes mediated by kalirin-7, whereas the broad deacetylase inhibitor NaBu does not exert such an effect. The asterisks indicate statistical significance (**P≤0.005). Error bars, S.E.
Figure 7.
An HDAC6 deacetylase-dead mutant opposes the formation of synphilin-1 containing aggresomes mediated by kalirin-7.
(A) HEK293 cells were triple-transfected with HcRed-synphilin-1, EGFP-kalirin-7 and FLAG-WT HDAC6 or FLAG-H216A/H611A mutant HDAC6. After 48 h cells were fixed and stained with anti-FLAG antibodies. Only cells co-expressing mutant HDAC6 form more cytoplasmic small aggregates (arrows). (B) Quantification shows that mutant HDAC6 inhibited the kalirin-7-mediated perinuclear synphilin-1 inclusion formation. The asterisks indicate statistical significance (***P≤0.001). Error bars, S.E.
Figure 8.
Decreased tubulin acetylation in kalirin-7 expressing cells under TSA treatment.
(A, B) An interaction of FLAG-kalirin-7, V5-synphilin-1 and HA-HDAC6 was examined by co-immunoprecipitation experiments. 24 h after transfection, HEK293 cells were lysed and 500 µg of protein lysates were subjected to immunoprecipitation with anti-FLAG or anti-V5 conjugated agarose beads, respectively. The precipitates were probed with HA antibodies to detect HDAC6 and revealed an interaction of HDAC6 with both kalirin-7 and synphilin-1. 30 µg of protein lysates were visualized as input control. The asterisk indicates a non-specific band observed in all raw lysates detected with anti-V5. (C) Cells transiently overexpressing FLAG-kalirin-7 (c, g), FLAG-kalirin-7 plus HcRed-synphilin-1 (d, h), or empty vectors (a, b, e, f) were immunostained for acetylated tubulin (a-d, green) and kalirin-7 (g, h light blue) after DMSO or 1 µM TSA treatment. While TSA treatment resulted in higher acetylation levels in comparison to controls (arrowheads), the overexpression of kalirin-7 led to a significant decrease of the α-tubulin acetylation levels (arrows). Blue, DAPI. Scale bar, 10 µm. (D) Acetylated tubulin levels were quantified by the fluorescence signal of individual cells, as described in Materials and Methods. Kalirin-7 transfected cells treated with TSA were compared to untransfected cells in the same cell population. Comparably, kalirin-7/synphilin-1 doubly transfected cells treated with TSA were quantified relative to untransfected cells in the same population. The asterisks indicate statistical significance (**P≤0.005). Error bars, S.E., n = 100.
Figure 9.
Proposed pathway of kalirin-7-mediated synphilin-1 aggresome formation.
(A) Under normal conditions, misfolded synphilin-1 is mainly accumulated in cytoplasmic small aggregates. (B) When kalirin-7 is overexpressed, it facilitates the recruitment of HDAC6 and the dynein motor complex and acts on microtubule dynamics by stimulating the deacetylase activity of HDAC6, thereby increasing the transportation of synphilin-1 into aggresomes.