Mammalian end binding proteins control persistent microtubule growth

Abstract

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.

Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes

“…5B), which was replaced by the punctuate pattern, suggesting that neoformed Ist2 clusters accumulate in intracellular ER sites and cannot be transported to the cell periphery. Because end-binding protein 1 (EB1) has recently emerged as a key regulator of dynamic +TIP interaction networks at growing microtubule ends (24,25), we investigated the consequences of EB1 silencing on TMD-Ist2ct trafficking. We used shRNAs (sh1-3) targeted against EB1 to down-regulate its expression.…”

Piecewise Constant Sequential Importance Sampling for Fast Particle Filtering

“…We study the performance of pcSIR by considering a biological image-processing application: the tracking of sub-cellular (here, "cell" refers to the biological cell being imaged and is not to be confused with the pcSIR bin cells) objects imaged by fluorescence microscopy [16][17][18]. There, intracellular structures such as endosomes, vesicles, mitochondria, or viruses are labeled with fluorescent dyes and imaged over time with a confocal microscope.…”

Control of Vascular Permeability by Atrial Natriuretic Peptide via a GEF-H1-dependent Mechanism

“…For microtubule quantification, cells were fixed with Ϫ20°C methanol, and immunostaining was carried out with ␤-tubulin or EB1 antibodies as described previously (25,26). Briefly, the cell boundaries were outlined by VE-cadherin staining, and concentric outline shapes reduced to 70% were applied to the images to mark peripheral (outer 30% of diameter) and central (inner 70% of diameter) regions.…”

“…EC were transfected with GFPtagged EB1, which tracks the growing plus-end of microtubules. EB1 tracking in live cells was performed as described previously (25,26). Projection images were generated as described under "Experimental Procedures."…”