Septin 6 localizes to microtubules in neuronal dendrites

Abstract

In neuronal dendrites, septins localize to the base of the spine, a unique position which is sandwiched between the microtubule (MT)-rich dendritic shaft and the actin filament-rich spine. Here, we provide evidence for the association of SEPT6 with MTs in cultured rat hippocampal neurons. In normal cultures, SEPT6 clusters localized to MTs, but not to actin clusters. Only MT-disrupting agents (vincristine and nocodazole), but not microfilament-disrupting one (latrunculin A), induced the redistribution of SEPT6 to the disrupted MTs. The nascent MT fibers that were recovered from vincristine or nocodazole treatments also accompanied SEPT6. Blocking MT disruption by Taxol prevented such phenomena, proving that the redistribution of SEPT6 was due to the MT disruption. Our results indicate that SEPT6 complexes at the base of the dendritic spine are associated with MTs.

Fig. 1

Disruption of actin filaments does not change the distribution profile of SEPT6. Confocal microscopic images showing dissociated rat hippocampal cultures (DIV 21), which were double-labeled with anti-SEPT6 and anti-pan actin antibodies. a Control culture. b Latrunculin A-treated (5 μM, 24 h) culture. c Recovered culture. Cultures were treated with Latrunculin A and recovered for 24 h in the control medium. The boxed area of a dendrite (box) was enlarged and shown at the bottom of each panel. Actin clusters and SEPT6 punctae are marked with arrowheads to show their relative positions neighboring each other. Note that there is no apparent change in the distribution profile of SEPT6 among different conditions. Scale bar 15 μm. d Statistics. The percentage of SEPT6 punctae that co-localize with MT was analyzed. The numbers of SEPT6 punctae counted [n = 143 (control), 112 (latrunculin), 196 (lat/recovered)]. **p < 0.01; *p < 0.05

Fig. 2

Disruption of MT fibers changes the distribution profile of SEPT6. Confocal microscopic images showing rat hippocampal dissociated cultures (DIV 21), which were double-labeled with anti-SEPT6 and anti-tubulin (Tub). a Control culture. b Vincristine treated (5 μM, 4 h) culture. c Nocodazole treated (30 μM, 2 h) culture. The boxed area of a dendrite was enlarged and shown at the bottom of each panel. Localization of SEPT6 punctae on MT fibers (a, c) or on tubulin paracrystals (b) are marked with arrowheads. Note that SEPT6 was changed into rod-shaped (arrows in b) or big clusters (arrowheads in c) in the presence of vincristine and nocodazole, respectively, but that they are still co-localized with MT fibers or paracrystals (merge images in b and c). Scale bar 15 μm. d Statistics. The percentage of SEPT6 punctae that co-localize with MT was analyzed. The numbers of SEPT6 punctae counted [n = 153 (control), 119 (vincristine), 146 (nocodazole)]. **p < 0.01

Fig. 3

Blocking of MT disruption prevents changes in the SEPT6 distribution profile. Confocal microscopic images showing dissociated rat hippocampal cultures (DIV 21), which were double-labeled with anti-SEPT6 and anti-tubulin (Tub). The cultures were pretreated with Taxol (10 μM, 15 min) to block MT disruption, followed by treatment with vincristine (a) or nocodazole (b). The boxed area was enlarged at the bottom of each panel. Note that SEPT6 punctae were lined up on MT fibers in preTax/vin (a). Due to less disruption of MT fibers in preTax/noc (b), SEPT6 punctae look randomly scattered. However, note that they locate to MT fibers. Statistical analysis is shown in d. The percentage of SEPT6 punctae that co-localize with MT was analyzed. Counted SEPT6 punctae: n = 78 (preTax/vin) and 142 (preTax/noc)

Fig. 4

Recovery from disruption re-localizes SEPT6 to the nascent MT fibers. a Confocal microscopic images showing dissociated rat hippocampal cultures (DIV 21), which were double-labeled with anti-SEPT6 and anti-tubulin (Tub). The cultures were treated with vincristine, washed and further incubated in the control medium for 48 h to recover MT fibers. The boxed area was enlarged at the bottom of each panel. Recovered MT-IR signals in the dendritic shaft was marked with an arrow (a Tub; inset), and nascent MT fibers with arrowheads (a Tub; inset). Note that SEPT6 punctae localize to the recovered MT fibers (merge). Scale bar 15 μm. b Statistics. The percentage of SEPT6 punctae (n = 104) that co-localize with MT was analyzed. Data for control and vincristine (vinc) of Fig. 2 were included for comparison. **p < 0.01