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. 2017 Sep 6;6:e26002. doi: 10.7554/eLife.26002

Figure 4. HA bath and a subsequent wash induced long-lived yet reversible changes to the MT system.

Figure 4.

(a) Static EB1 signals remained at BBs after cells were resuspended in 10 mM pH3 HA for 5 min (left panel). After replacing HA with the HEPES buffer, EB1 signal at the BB area intensified within 1 min (black arrowheads). But newly formed MTs were thick and prominent, lacking the typical comet (right panel, cell I). In cells recorded 60 s after wash (cell II and III), MT elongation slowed down gradually. (b) In cells imaged ~180 s after wash, EB1-decorated MTs in all cells stopped growing (top panel), as highlighted in two additional images of two representative cells captured 20 s apart. In addition, a MT fiber in cell I split into two (arrowhead), while a fiber in cell II had multiple comets aligned in tandem (arrowheads), as if new MTs nucleated or grew on older ones. (c) Kymographs comparing the growth of three representative MTs in cells pretreated with 5 min HA bath and then the wash buffer (top panels) and in control cells in the HEPES wash buffer (lower panels). Comets in the latter manifested as an intense spot at the plus end. The sharper slopes in the former indicated slower growth. Unchanged slopes indicated paused growth. (d) Long-lived MTs formed after HA bath and wash were cold resistant (top panels). As shown in two representative cells, frozen MTs remained in the image captured 20 s after 3 min on ice. The changes in subsequent images were due to focal plane drifts as cells were floating gradually as warming up. In control cells without previous HA exposure (bottom panels), EB1 signals were absent initially (20 s), consistent with cold lability. Dynamic comets (white arrowheads) gradually emerged at the BB area afterwards. (e) Although the MT system froze within minutes after HA bath and HEPES wash (t = 0 min), dynamic comets resumed after 55 mins in cells recovered in TAP media as shown in two images captured 20 s apart. Scale bars, 5 μm. (f) A schematic summarizing the sequence of MT changes induced by HA bath and wash.