Detectability of endocytosed NgCAM in somatodendritic endosomes depends on permeabilization conditions
Because it remains controversial whether or not NgCAM is found in somatodendritic endosomes (Sampo et al., 2003
; Wisco et al., 2003
), we first tested what variables might affect detection of endocytosed NgCAM in somatodendritic endosomes. Neither the transfection method nor the age of the culture showed any differences in the detectability of somatodendritic NgCAM endocytosis (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1
). Detergent conditions, however, proved critical for reliable detection of endocytosed NgCAM (Fig. S1, B–E). The discrepancy between the findings of Sampo et al. (2003)
and our own (Wisco et al., 2003
) therefore likely reflects differences in permeabilization conditions. Similarly to NgCAM, human L1-myc (Fig. S1 F) and rat L1-myc (not depicted) were internalized into somatodendritic endosomes. Cross linking of NgCAM by divalent anti-NgCAM IgGs is not necessary for the detectability of NgCAM in somatodendritic endosomes because somatodendritic endocytosis can also be observed with comparable efficiency (i.e., 70–80% of cells) when anti-NgCAM Fab fragments are used for the endocytosis assay (Fig. S1 G).
Endocytosed NgCAM colocalizes with somatodendritic EEs and partially localizes with lysosomes
The selective retrieval/retention model predicts that endocytosed NgCAM would be primarily degraded (, red), whereas the transcytotic model predicts that NgCAM would largely bypass the late endosome (LE)/lys (, blue). We, therefore, asked whether endocytosed NgCAM in the somatodendritic domain initially colocalized with a marker for EEs (EEA1), and whether or not it ultimately accumulated with a marker for LE/lys (lgp120; see Materials and Methods for details on quantification). After incubating NgCAM-expressing cells for 20 min with anti-NgCAM antibodies (t = 0), 55% of all labeled endosomes contained both endocytosed NgCAM and EEA1 (, yellow, orange, and light green segments on the left), 23% of all labeled endosomes contained only EEA1 (, red; and , left, red). If only EEA1-positive endosomes were analyzed, 71% of them contained some endocytosed NgCAM (“high” plus “low” colocalization categories). We currently do not know if the high and low colocalization endosomes represent functionally distinct endosomal populations but they might reflect progressive sorting of NgCAM out of EEA1-positive endosomes.
Endocytosed NgCAM, which had been chased for 1 h (t = 60) to allow accumulation in a terminal compartment, colocalized partially with lgp120 (21% of all labeled endosomes; , yellow; and , right, yellow), whereas 32% contained only endocytosed NgCAM (, right, red). Of the somatic endosomes containing endocytosed NgCAM at long chase times, 47% also contained various amounts of lgp120. Because ~50% of endocytosed NgCAM leaves the somatodendritic endosomes in 90 min (see ), we estimate that ~25% of endocytosed NgCAM ultimately accumulates in lysosomes. Because all proteins turn over to some degree at all times, some NgCAM is expected to localize to lysosomes. We note that complete quantification of NgCAM localization to lysosomes is not possible because some NgCAM and bound antibody might have been already degraded.
Figure 3. Endocytosed NgCAM leaves somatic endosomes and recycles preferentially to the axonal plasma membrane. (A) After 20 min of anti-NgCAM antibody loading (t = 0), intracellular endocytosed NgCAM can be visualized after acid stripping of surface-bound (more ...)
Somatodendritic endosomes contain endocytosed NgCAM by immuno-EM
Next, we sought to identify the somatic compartments containing NgCAM on the ultrastructural level. NgCAM at steady-state was found prominently in multivesicular bodies (MVBs; , asterisks) in the soma. Small profiles (, arrowheads) were also labeled. The exact identity of these compartments is not known but they might correspond to transport carriers or tubular portions of EEs.
Figure 2. Ultrastructural identification of NgCAM-containing somatic endosomes. (A) Hippocampal neurons were infected with AdNgCAM for 24 h and subsequently fixed. NgCAM was detected with anti-NgCAM antibodies and 15 nm gold–protein A. NgCAM is found on (more ...)
To visualize endocytosed NgCAM, neurons were incubated simultaneously with an anti-NgCAM antibody and the fluid-phase marker gold-BSA and then processed for immuno-EM (Oorschot et al., 2002
). Gold-BSA is ultimately transported to lysosomes and does not accumulate in REs. We therefore determined if endocytosed NgCAM could accumulate in compartments that exclude gold-BSA. Endocytosed NgCAM, detected by 15-nm gold-coupled protein A, was most often found in MVBs together with 3-nm gold-BSA (). Some of these NgCAM-positive MVBs contained no or very little gold-BSA (). 15 nm gold was also occasionally associated with tubules devoid of gold-BSA, which is reminiscent of recycling endosomal compartments (; Peden et al., 2004
). NgCAM is therefore capable of entering nondegradative endosomal compartments including REs, but is also found in presumptive predegradative compartments.
Endocytosed NgCAM traverses somatodendritic endosomes and recycles preferentially to the axonal plasma membrane
The fate of endocytosed NgCAM was followed using a pulse-chase approach with an acid strip. We incubated NgCAM-expressing neurons with an anti-NgCAM antibody for 20 min (load) and then removed all remaining surface-bound antibody using an acid strip step. Cells were then returned to the incubator for various amounts of time (chase). After loading (t = 0), endocytosed NgCAM could be detected prominently in somatodendritic endosomes () and was also seen, though less brightly, in endosomes along axons (, arrows). After 90 min of chase, the soma fluorescence was decreased (), whereas axonal endosomes were still bright (, arrows). Quantification of the soma-associated fluorescence showed that the half-time for depleting the soma signal was ~90 min (, open diamonds). Tf, however, disappeared from the soma with a half-time of ~25 min (, circles). To estimate the abundance of endocytosed NgCAM within dendritic and axonal endosomes, we quantified the intensity of individual endosomes in axons and dendrites. Endosomes correspond to the peaks in intensity line scans () taken along dendrites and axons after the loading step (t = 0′) and at t = 90′. At t = 0, dendritic endosomes were on average about twofold brighter than axonal endosomes (, top; axon/dendrite intensity ratio = 0.58). At t = 90′, axonal endosomes were on average 40% brighter than dendritic endosomes ( bottom; axon/dendrite intensity ratio = 1.39). Endocytosed NgCAM, therefore, progressively disappeared from somatodendritic endosomes and accumulated in axonal endosomes.
To see if internalized NgCAM ever reappeared on the cell surface, we performed a load/acid strip/chase experiment as described in the previous paragraph but incubated the cells with Alexa 647–coupled secondary anti–mouse antibodies before permeabilization to stain the recycled surface pool (see Materials and methods). Initially, no surface labeling could be detected (′), but surface reappearance was easily detected at t = 90′ (, triangles and broken line). To determine to which surface endocytosed NgCAM recycled, we determined the axon-dendrite polarity index (A/D PI; see Materials and methods) for recycling NgCAM. At t = 40′, surface NgCAM staining already was threefold higher on the axonal surface. By t = 90′, it was 4.7-fold higher. By 2.5 h of chase, the recycling A/D PI was 5.5, the same as the steady-state A/D PI for NgCAM. NgCAM therefore recycled with at least a threefold bias toward the axon.
Endocytosed NgCAM accumulates in stationary somatodendritic endosomes and is transported in small endosomal carriers
We next imaged trafficking of endosomal NgCAM by feeding anti-NgCAM antibodies coupled to Alexa 488 to NgCAM-expressing neurons for 1 h in a live-imaging chamber. Cells were then washed and images were taken every 2 or 3 s for about 1 min. Endocytosed NgCAM was easily detected in somatodendritic endosomes in live cells (). The extent of endosome motility was displayed by merging three video frames into one RGB image such that the first frame appeared red, the second green, and the third blue (). Interestingly, the majority of the bright endosomes in the soma were stationary during the imaging duration (; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1
). A subset of NgCAM-containing endosomes underwent movements (, arrowheads). The labeling intensity of the endosomes usually correlated with size, such that the large stationary endosomes were brightly labeled but the small moving carriers were faint. We frequently observed short, wiggling excursions that did not result in long-distance translocation. We also observed longer-range transport events of small tubules (, arrows) or small circular compartments (, arrows). Movements were often intermittent with pauses of many seconds interspersed with periods of movement. Frequently, a small transport carrier initially appeared to emerge from, and ultimately disappear into, a larger stationary endosome. 91% of NgCAM-containing endosomes in the soma and dendrites appeared to be round or irregularly shaped, whereas 9% appeared to be elongated (n
= 232; ). The elongated carriers frequently appeared to be of varying width and intensity along their lengths (see Sonnichsen et al., 2000
). 22% of all observed NgCAM endosomes underwent movement during the 1-min imaging periods. Of all the moving compartments, 84% appeared small (estimated diameter of ~0.2 μm) and round, whereas 14% appeared elongated. Large (estimated diameter, >1 μm) and medium-sized (estimated diameter, ~0.7 μm) round compartments rarely moved (, white and black bars). NgCAM therefore accumulates in stationary somatodendritic endosomes but is transported intermittently in small transport carriers.
Figure 4. Live imaging of NgCAM-containing endosomes. (A–C) Neurons (DIV10) transfected with NgCAM were allowed to endocytose Alexa 488–anti-NgCAM antibodies in live imaging chambers for 60 min before washing and imaging at an acquisition speed (more ...)
Endocytosed NgCAM travels anterogradely in axons
Retrograde transport of endosomes has been well described (Overly and Hollenbeck, 1996
; Segal, 2003
; Deinhardt and Schiavo, 2005
). Whether or not endosomal cargo travels anterogradely in axons has not been established. We therefore sought to determine the mobility of NgCAM endosomes in the axon (see Materials and methods). Again, most of the bright round endosomes containing NgCAM were not motile (, white puncta). Because the moving endosomal carriers are small and faint, imaging of axonal motile endosomes was technically challenging and extensive quantification proved not to be feasible. Nonetheless, we could observe clear examples of moving transport carriers containing endocytosed NgCAM ( and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1
) in both the anterograde and retrograde direction (, arrows; and Video 2) with speeds ranging from 0.3 to 1.8 μm/s. Slightly more than half of the movements occurred in the anterograde direction. Therefore, small NgCAM-containing endosomal carriers travel up the axon by anterograde axonal transport, whereas large stationary endosomes in the axon accumulate endocytosed NgCAM. We do not currently know whether the retrogradely transported NgCAM is degraded in lysosomes or can recycle to the plasma membrane.
Endocytosed NgCAM transiently colocalizes with recycling Tf
Tf is a marker for the somatodendritic recycling pathway (, green; Hemar et al., 1997
). The transcytotic model predicts that NgCAM initially internalizes into the same EE as Tf but ultimately sorts away from Tf into distinct endosomes and is sorted to axons from there. We incubated FITC-Tf–loaded cells with anti-NgCAM antibody at 16°C for 30 min to allow entry into the EE (Schmid and Smythe, 1991
). Cells were then washed and incubated at 37°C in the continued presence of FITC-Tf for chase times of 5–120 min. At 5 min of NgCAM chase, NgCAM and Tf showed numerous clear examples of precise overlap (unpublished data), suggesting that NgCAM initially entered the Tf-containing EE, which is consistent with the colocalization of EEA1 and endocytosed NgCAM (). At 15 min of chase, we already observed many puncta containing only endocytosed NgCAM (, left, red). Because Tf was loaded to a steady state, these puncta correspond to a compartment in which Tf does not significantly accumulate. Precise colocalization of Tf and endocytosed NgCAM at 15 min of chase could also be observed (, arrowheads). After chase times of 60 min (, right), Tf- and NgCAM-positive regions frequently appeared closely apposed but not precisely overlapping. In these cases, the red and green channels were laterally offset, appearing as multicolored “traffic lights” (, right, arrowheads), which suggests that Tf and NgCAM largely occupied either distinct compartments or distinct domains of the same compartment (see also Thompson et al., 2007
). This pattern of localization suggested that NgCAM and Tf entered the same EEA1-positive EE and subsequently sorted away from one another in a somatodendritic endosomal compartment, presumably an RE.
Figure 5. Dual live imaging of internalized Tf and NgCAM. (A) Neurons were transfected with NgCAM and incubated with anti-NgCAM antibodies (red) and FITC-Tf (green) at 16°C, washed and chased at 37°C in the continued presence of Tf for 15 (A, left) (more ...)
Dual live imaging of endocytosed NgCAM and Tf
We next imaged NgCAM-expressing cells loaded with Texas red–Tf and Alexa 488–coupled anti-NgCAM antibodies to see if Tf and NgCAM were transported together in endosomal carriers (see Materials and methods). The Tf-positive compartments correspond to REs because they still contained Tf after >20 min of chase. We observed somatodendritic endosomes containing both (, yellow), or either one of the cargos (, green and red). 42% of endosomes contained both NgCAM and Tf, 30% contained only NgCAM, and 28% contained only Tf (n
= 365). demonstrates some of the classes of behaviors we observed: arrowheads designate some of the stationary endosomes, whereas arrows designate motile endosomes (Vidoes 3–6, available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1
). Of all labeled endosomes (n
= 365), 17% underwent movements in 50 s of imaging. Tf-containing compartments frequently wiggled about randomly, but only a few examples of rapid directed movement of Tf-containing compartments could be observed (, red arrow; and , right). Many of the compartments containing only Tf were large and pleiomorphic. Shape changes in these large structures did not permit quantification of individual compartments. In contrast, we frequently saw clear examples of mobile small spherical and tubular elements. These motile compartments tended to be largely devoid of Tf, with 70% of mobile compartments containing only NgCAM (, middle; and Video 3). Small spherical endosomes showed the most motility (, green arrow; , middle; and Video 4).
In addition, we observed red and green puncta that moved as a unit ( and Video 5), which is reminiscent of the traffic light patterns observed in fixed samples (, right). These images might represent single membrane-bound elongated compartments in which Tf and NgCAM are laterally segregated. Such lateral segregation of cargos in REs is also found in other cell types, including MDCK cells (Thompson et al., 2007
) and macrophages (Manderson et al., 2007
). To visualize the laterally segregated cargos, a fluorescent lipid (DiIC18-DS) was allowed to coendocytose. This assay enabled the observation of two differently labeled cargos encompassed in single DiI-labeled structures (Manderson et al., 2007
). Even though this technique still relies on light microscopy, observation of such structures is consistent with the existence of single compartments with laterally segregated components. We adapted this assay, feeding Tf and anti-NgCAM antibody as well as DiI. Surprisingly, DiI labeled the NgCAM-containing endosomes much more reliably than it labeled Tf-containing endosomes (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1
), which suggests that the lipid composition of NgCAM and Tf endosomes is not identical and that DiI partitions preferentially into a subset of endomembranes in neurons. We occasionally observed endosomes labeled by all three tracers (two to five per cell out of hundreds). Among the triple-labeled profiles, endosomes with laterally offset NgCAM and Tf staining seemingly encompassed by a single DiI-labeled profile were common (Fig. S3, B–D). Endocytosed NgCAM therefore was found together with Tf in stationary REs in the soma but it segregated away from Tf and moved over long distances in small spherical or tubular endosomal carriers largely devoid of Tf.
Axonal polarization of NgCAM is sensitive to interference with NEEP21 but not toxin-insensitive vesicle-associated membrane protein (Ti-VAMP)
In PC12 cells, two proteins affect the endosomal recycling of L1, the toxin-insensitive VAMP Ti-VAMP/VAMP7 (Martinez-Arca et al., 2001
) and NEEP21 (Steiner et al., 2002
). Ti-VAMP is found in both somatodendritic and axonal endosomes, whereas NEEP21 is found only in somatodendritic endosomes. We used two function-interfering constructs, Ti-VAMP-DN (Martinez-Arca et al., 2001
) and antisense NEEP21-GFP (AS-NEEP21; see Materials and methods for details; Steiner et al., 2002
). GFP was cotransfected with NgCAM as a control. Coexpression of Ti-VAMP-DN did not significantly affect A/D PI for NgCAM (, left), even though it profoundly inhibited axon outgrowth if introduced at day in vitro (DIV) 2 (not depicted; Martinez-Arca et al., 2001
). Coexpression of AS-NEEP21, however, led to missorting of NgCAM to dendrites, resulting in a statistically significant reduction of NgCAM A/D PI (, right). NEEP21 is therefore a regulator of NgCAM transport in hippocampal neurons.
Figure 6. Endocytosed NgCAM traverses the NEEP21-positive EE. (A) Neurons were cotransfected with NgCAM and either dominant-negative Ti-VAMP (left) or anti-sense NEEP21 (right). GFP was expressed as a control. 18 h after transfection, surface NgCAM was detected (more ...)
Endocytosed NgCAM (red) substantially and precisely overlapped with endogenous NEEP21 (, green) in somatodendritic endosomes (66% of all labeled endosomes; , left). Of NEEP21-containing endosomes, 86% contained endocytosed NgCAM at t = 0. At 1 h, the percentage of all labeled endosomes containing both NEEP21 and endocytosed NgCAM fell to 48% (, right). The NEEP21 compartment, therefore, behaves kinetically as an intermediate on the NgCAM pathway. Interestingly, the rate of clearance of endocytosed NgCAM from NEEP21 endosomes is slow, indicating that NgCAM has a long residence time in this compartment. Substantial colocalization with NEEP21 was also observed for endocytosed human and rat myc-L1 (not depicted). In contrast, NEEP21 (, green) showed only poor overlap with EEA1-positive EEs (red) in cultured hippocampal neurons (), as is the case in PC12 cells (Steiner et al., 2002
Down-regulation of NEEP21 causes missorting of NgCAM in endosomes
Increased missorting to the somatodendritic domain could arise by several mechanisms. NgCAM might be expressed at higher levels in AS-NEEP21–expressing cells, leading to saturation of the sorting machinery. We therefore compared the total mean surface intensity of NgCAM in cells expressing GFP as control or AS-NEEP21. No statistically significant differences in total surface NgCAM were observed (surface intensity in GFP = 5208 ± 832 arbitrary units; AS-NEEP21 = 5952 ± 534 arbitrary units). Because NEEP21 is found in the TGN as well as in endosomes (Steiner et al., 2002
), either one could be the site of missorting. We therefore determined the A/D PI at steady state of a mutant NgCAM containing a point mutation in the somatodendritic sorting signal, NgCAM Y33A. This NgCAM mutant travels to the axon on a direct pathway from the TGN, bypassing somatodendritic endosomes (Wisco et al., 2003
). The steady-state A/D PI of NgCAM Y33A was not reduced by coexpressing AS-NEEP21 (). Additionally, we determined the A/D PI of endosomally recycling NgCAM (using the same assays as for ). The recycling A/D PI of NgCAM was also significantly diminished compared with control cells (, right). AS-NEEP21 therefore causes missorting of NgCAM during endosomal recycling.
Figure 7. Down-regulation of NEEP21 leads to missorting of endosomal NgCAM to the somatodendritic surface and to lysosomes. (A) The A/D PI was determined for NgCAMY33A at steady state (left) and for NgCAM for endosomal recycling to the plasma membrane (t = (more ...)
NgCAM missorting occurs in the EE
We next asked whether the kinetics of endosomal trafficking of NgCAM were changed in AS-NEEP21–expressing cells. We predicted that endocytic uptake of NgCAM would be increased because higher levels of NgCAM are available for endocytosis on the somatodendritic surface at steady state in AS-NEEP21–expressing cells. We do in fact see more than twofold higher levels of endocytosed NgCAM in the soma at t = 0 in AS-NEEP21 compared with GFP controls (). For several other receptors, expression of AS-NEEP21 caused increased retention of the receptors in endosomes. We therefore compared the extent of NgCAM clearance from somatic endosomes at t = 40 min. Signal levels at t = 40′ were normalized to signal levels at t = 0 for each set because the initial signal levels were much higher for AS-NEEP21 to begin with (). We found a modest increase in NgCAM retention in AS-NEEP21–expressing cells (P = 0.055 by Mann Whitney U test).
In epithelial cells, cargo can leave the EE in three directions: direct local recycling to the plasma membrane, transport to the RE, or transport to the LE/lysosome (see ). We next asked if the retention of endocytosed NgCAM in somatic endosomes () might be, at least in part, caused by terminal missorting to LE/lysosomes. In control cells (, top), little overlap between NgCAM (red) and the lysosomal lgp120 (blue or aqua in insets) was observed (overlap appears white in the insets). AS-NEEP21–coexpressing cells, however, showed increased colocalization of endocytosed NgCAM with lgp120 (, bottom; and ). NEEP21 function therefore biases the exit route from the EE such that NgCAM is preferentially transported to the RE rather than recycled to the somatodendritic surface or trafficked to lysosomes.
Lastly, we asked if Tf was missorted to the axon in AS-NEEP21–expressing cells. Such missorting would suggest that the somatodendritic EE could serve as a direct exit station toward the axon. No such missorting was observed and Tf remained somatodendritically restricted in antisense NEEP21–expressing cells (, aqua), whereas endocytosed NgCAM was additionally found in axonal endosomes (, red; arrows indicate axons).
Polarity of endogenous L1 is affected by down-regulation of NEEP21
We then asked if the axonal polarity of endogenous L1 was also dependent on NEEP21 function. We introduced the antisense NEEP21 plasmid by electroporation into dissociated hippocampal neurons before plating and then assayed the distribution of surface L1 at DIV3. Endogenous L1 was highly enriched on the axonal surface (, arrows) in GFP-expressing control cells (, red) and little L1 was detected on the somatodendritic surface (arrowheads). When AS-NEEP21-GFP was expressed, L1 was readily detected on the axon (, arrows) but also on soma and dendrites (, red, arrowheads). The A/D PI of endogenous L1 was significantly reduced in antisense NEEP21–expressing cells (). NEEP21 therefore promoted axonal polarization of endogenous L1 in young cultures. Because of two technical constraints, we were unable to assess the effects of AS-NEEP21 on endogenous L1 in mature cultures. (1) Endogenous L1 accumulates stably to high levels on the axonal surface. Introducing antisense NEEP21 at DIV8/9 (as we do for exogenously expressed NgCAM) made it impossible to detect the fate of the newly synthesized pool of L1 because such a large amount of endogenous L1 was already accumulated stably on the axonal surface at the time of transfection. (2) Mature cultures of DIV9–11 have a very dense network of criss-crossing axons. Trying to assess the polarity of endogenous L1 proved not to be possible because individual axons cannot be unambiguously traced to the cell body. Furthermore, axons commonly grow along dendrites, giving the erroneous impression that MAP2-positive processes are L1-positive.
Figure 8. Endogenous L1 mislocalizes to the somatodendritic domain when NEEP21 is down-regulated. (A and B) Dissociated neurons were electroporated with GFP as control (A and A′) or AS-NEEP21-GFP (B and B′) and surface L1 was detected with a polyclonal (more ...)
The observation that endogenous L1 is sensitive to NEEP21 depletion suggested that a significant fraction of endogenous L1 also travels via somatodendritic endosomes to the axon. We therefore tested if endogenous L1 can be endocytosed in the somatodendritic domain by performing antibody uptake experiments in DIV3 neurons with an antibody against the endogenous L1. We observed extensive labeling of endosomes, both in axons and in the somatodendritic domain ().
Live imaging of NEEP21-GFP and endocytosed NgCAM
Lastly, we determined if the NEEP21 compartment corresponded to the stationary NgCAM-containing endosomes or whether NEEP21 was also found in moving transport carriers together with NgCAM ( and Videos 6 and 7, available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1
). Colocalization of endocytosed NgCAM and NEEP21-GFP was striking and precise (, yellow). These yellow compartments containing both NgCAM and NEEP21 were largely stationary (, asterisk), with only 2.5% of them moving (; n
= 443). Unexpectedly, NEEP21-containing compartments did show frequent movements (, arrowheads) but the moving NEEP21 compartments rarely contained NgCAM. If only moving compartments were considered, 66% of them contained only NEEP21 (). Compartments containing NgCAM but not NEEP21 also moved (20.5%; , white arrows; and ). The observed movements were episodic, frequently punctuated by pauses of many seconds (). We conclude that NgCAM accumulates in stationary NEEP21 endosomes and is transported in NEEP21-negative transport carriers. NEEP21-positive carriers are frequently observed but it is not known which cargo might be cotransported.
Figure 9. Dynamic behavior of NEEP21-GFP and endocytosed NgCAM in endosomes. (A and B) Live imaging of endocytosed NgCAM (red) and NEEP21-GFP (green). A portion of a proximal dendrite is shown (A). Images were captured every 2 s as indicated above each panel. Frames (more ...)