Fig 1.
SARA subcellular distribution in young cultured hippocampal pyramidal neurons.
(A) Confocal image showing an example of stage 2 neurons (1DIV) or (B) stage 3 neurons (2-3DIV) immunostained for SARA (green). Note that SARA immunolabeling is present in the soma, in all types of neurites (minor process and axon) and that it extends into neuritic tips. SARA is associated with early and recycling endosomal compartments. (C-H) Neurons double-labeled, using antibodies against EEA1 (green, C) and SARA (red, D) or Rab11 (green, F) and SARA (red, G). The SARA-labeled punctae displayed partial colocalization with the early endosomal marker (E, inset) or recycling endosomal marker (H, inset). Different heterogeneous populations of endosomes were observed: one containing only EEA1 or Rab11, a second with EEA1 or RAb11 and SARA, and a third containing only SARA endosomes. All cells were imaged by confocal microscopy. Data is typical of over 10–20 neurons imaged for each condition.
Fig 2.
Characterization of Rabs distribution in neurons with modified SARA expression.
Hippocampal pyramidal neurons at 7DIV were transfected with (A) Rab5-GFP, (F) Rab4-GFP or (K) Rab11-GFP where usual Rabs localization was observed. In neurons cotransfected with Rab5-GFP + SARA-Flag (B-C, inset), Rab5 labeling was extensively colocalized with SARA-Flag on enlarged early endosomal membranes; (G-H) no obvious alterations were observed for Rab4 endosomes after SARA overexpression. (L-M) Images showing intracellular distribution of Rab11-positive endosomes when SARA was overexpressed. Images of neurons transfected with (I-J) Rab4-GFP or (N-O) Rab11-GFP and cotransfected with sh-SARA (red); Rab4- and Rab11-positive recycling endosomes were distributed occupying the neuronal perikaryon area compartment compared to their distribution in controls (I vs F; N vs K). (D-E) No changes were observed in Rab5-GFP neurons cotransfected with sh-SARA. This suggests that there was a change in the Rab4-positive and Rab11-positive vesicular compartments in EE and RE after SARA suppression.
Fig 3.
Rab11 and Rab4 positive endosome distribution in SARA-KO neurons.
(A-D) Double-stained neurons for Rab11 and Tyr-Tubulin. Tyr-Tubulin labeling is used to show the general morphology of the neuronal cells. (E-H) Double-stained neurons for Rab4 and Rhodamine–Phalloidin. Phalloidin stains F-actin and shows integrity of actin cytoskeleton. Note that Rab11- and Rab4-positive recycling endosomes appear as a scattered fluorescent signal throughout the neuronal soma in SARA-KO neurons (C, G) vs juxtanuclear localization in control neurons (A, E). All cells were imaged by confocal microscopy. Data is typical of over 10 neurons imaged for each condition.
Fig 4.
SARA suppression enhances axon elongation and induces formation of supernumerary axons.
(Upper panel, left) Morphology of 1 DIV neurons transfected with GFP (A) or SARA-GFP (C), double stained with a mAb against Tyr-Tubulin (B and D). Note that neurons with overexpressed SARA have their neuritic development arrested or delayed. (Upper panel, right) Images showing examples of neurons treated with control sh-scr-HcRed (control; E-F) or sh-SARA (G-H). Cultures were transfected with the corresponding plasmids (1–2 μg DNA each) 24 h after plating and counterstained with a mAb against the axonal marker Tau-1 (green, F-H). Note that SARA suppression increases axonal length and produces the formation of supernumerary axons (arrows in H), contrasting with the single axon in control neurons (arrow in F). Quantification of neurite length in knockdown SARA neurons reveals a significant increase in minor processes, axons and dendrites (**p<0.001; R), respect to the control or SARA overexpressed neurons. For dendrite measurement, neurons were transfected with the same constructs but at 7 DIV and counterstained with a mAb against MAP2, a marker of neuronal dendrites. (Bottom panel) Hippocampal pyramidal neurons from control mice (I-K) or SARA-KO mice (L-Q) fixed at 1, 2 or 3 DIV and stained with a mAb against Tau-1 (green) and Rhodamine-Phalloidin (red). Note that the most of SARA-KO neurons show two or more axons (arrows). Confocal images were from independent experiments, litter mice, and different pregnant females. Bar graph shows average number of Tau-1-positive axons / 50 neurons analyzed (**p<0.001; S).
Table 1.
Effect of SARA suppression or overexpression on neuronal shape parameters.
Fig 5.
SARA as regulator of membrane delivery.
(Upper panel) Images from FRAP experiments in CHO-K1 cells. RE was photobleached in the regions of interest (ROI, circles) in cells expressing Rab11-GFP with sh-scr-HcRed (A) or co-transfected with sh-SARA (B). Arrows indicate Rab11-positive recycling endosomes dispersed over all cells. Recovery of fluorescent molecules from the surrounding area into the photobleached region was monitored over the 150 sec during which the frames were taken. The graph quantifies the recovery into the photobleached area as the fluorescence intensity obtained in three different FRAP experiments (5 control or sh-SARA cells were analyzed in each experiment). SARA suppression significantly increases the movement of Rab11-GFP endosomes into photobleached areas (C). (Bottom panel) Suppression of SARA alters the surface distribution of the axonal membrane protein L1. Confocal images showing the surface distribution of L1: (D-E) control neurons stained for Tyr-Tubulin or transfected with: (F-G) HcRed, (H-K) sh-SARA, (L-M) SARA-Flag. Neurons were exposed to primary antibody against L1 without permeabilization. (H-K) Images show apparent increase of L1 in the axonal membrane compared to the L1 level in control or SARA over-expressed neurons. Quantification of fluorescence intensity (IF) of 3 different regions of the axons (L = 10 μm each region) and the IF/L ratios were averaged to obtain a single value by neuron (n = 12 control and sh-SARA neurons). The graph confirms the observation finding more L1 on the axonal surface of SARA silenced neurons (N) (**p<0.001).
Fig 6.
SARA suppression perturbs somatodendritic protein delivery.
Time lapse images by TIRFM showing fusion events of vesicles to the plasma membrane in neurons of 7 DIV, transfected with TfR-GFP plus HcRed (A) or SARA (B). Images were taken every 2 seconds for at least 2 minutes. The graphic shows a significant reduction of fusion events at the somatodendritic plasma membrane in cells transfected with SARA plus TfR-GFP and significantly more fusion events in sh-SARA transfected neurons (C; n = 17 neurons; **p<0.001). TheTfR-GFP cell-surface protein increases after SARA silencing. CHO-K1 were transfected with TfR-GFP alone or plus SARA-Flag or sh-SARA. The cultures were incubated with the membrane-impermeable sulfo-NHS-biotin at 4°C to prevent endocytosis. Then the cells were harvested and biotinylated proteins were collected with avidin-agarose beads (Pierce). Membrane (D) and intracellular (E) proteins were isolated by centrifugation and analyzed by Western Blot with the indicated antibodies. Absence of cytosolic contamination was corroborated with anti-LIMK1 antibody. Graph showing the increase of TfR-GFP fluorescence intensity relative to total protein load (labeled with Tubulin; F) in SARA suppressed neurons respect to control or SARA overexpressed neurons. Data are means of three independent experiments.