Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin

Abstract

The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at the plus- or minus-ends of the polymer, is essential for MTs to carry out their biological functions. A variety of proteins act on MT ends to regulate their dynamics, including a recently described family of MT minus-end binding proteins called calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin/Nezha. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minus-ends against depolymerization in vitro and in vivo. Here, we show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. By using truncation analysis, we find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, we find that, in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics.

Keywords: TIRF microscopy; cytoskeletal regulation; tubulin polymerization.

Fig. 1.

CAMSAPs have a conserved function of binding MT minus-ends. (A) Most vertebrate species possess three CAMSAP/Patronin homologs, whereas Drosophila and C. elegans each have only one. (B) Motor gliding assay for determining the end of the MT bound by CAMSAPs; in a kinesin gliding assay, the minus-end of the GMPCPP-stabilized MT is the leading end; polarity is reversed in a dynein gliding assay. Kinesin assay for GFP-CAMSAP2 is shown. Arrows show GFP-CAMSAP2 on MT minus-ends. (Scale bar: 5 μm.) Movement of an MT–CAMSAP2 complex over time (Scale bar: Inset, 2 μm.) (C) Quantification of number of MT with GFP-CAMSAPs on their ends. Results compiled from two independent experiments (>100 MTs scored per assay).

Fig. 2.

CAMSAPs differentially affect tubulin subunit addition at MT minus-ends. (A) GMPCPP seed MTs (with Alexa-647 tubulin and biotin tubulin) were attached to an anti-biotin antibody–coated coverslip (Materials and Methods). Unpolymerized tubulin (23 μM) was added to the chamber with Alexa 561-labeled tubulin (2 μM) and allowed to polymerize at room temperature. (B) Control field of polymerizing MT. GMPCPP seeds shown in blue and newly polymerized tubulin are shown in red. Image was background-corrected in Fiji (SI Materials and Materials). Image shown ∼3–4 min after reaction setup. (Scale bar: 5 μm.) (C) Dynamic assay performed with addition of 10–12 nM GFP-CAMSAP3 (green spot at MT minus-ends) shows little growth after 3–4 min of polymerization. (Scale bar: 5 μm.) (D) Representative kymographs showing polymerization of control MT and MTs bound by GFP-CAMSAPs. Photobleaching of CAMSAP2 was partially corrected by using Fiji plugin (SI Materials and Materials). Minus-ends are oriented to the left. (Scale bars: horizontal, 1 μm; vertical, 20 s.) (E) Quantification of MT minus-ends showing visible growth in <4-min rapid acquisition window (control, n = 4; CAMSAP1, n = 2; CAMSAP2, n = 3; CAMSAP3, n = 4, where n is number of experimental days; n = 19–138 MTs per day). Minus-ends were scored as dynamic if they underwent growth of >0.5 µm over the course of imaging (200 s). Mean and SEM are shown for averages of each experimental day. Only MTs with GFP at the minus-end were scored. (F) Long-term (20 min) imaging of MT seeds (blue), newly polymerized tubulin (red), and GFP-CAMSAP2 and -3 (green). Minus-ends are oriented to the left. (Scale bar: 1 μm.) (G) Quantification of MT plus- and minus-end growth rates; short-term imaging (<4 min) was measured for plus- and minus-end growth rates; long-term imaging was used for CAMSAP2 and CAMSAP3 minus-ends. Number of independent experiments on separate days are as follows. Plus ends: control, n = 6; CAMSAP1, n = 2; CAMSAP2, n = 5; CAMSAP3, n = 5. Minus-ends: control, n = 6; CAMSAP1, n = 2; CAMSAP2, n = 2; CAMSAP3, n = 2, for n = 19–77 MTs per day. Mean and SEM of independent days of measurement are shown.

Fig. 3.

CAMSAP domains involved in minus-end MT binding and regulation of dynamics. (A) CAMSAP domain structure; predicted CC regions shown as cylinders. (B) The CKK domain of CAMSAP3 (20 nM) binds along the length of MTs with an accumulation at MT minus-ends, as shown by TIRF microscopy. CKK binding is abolished in the presence of 60 mM KCl. CAMSAP3 CC does not exhibit MT binding, but the CC+CKK domain shows specific MT minus-end binding in the presence of 60 mM KCl. (Scale bar: 2 μm.) (C) Representative kymographs of MTs capped by the CAMSAP2 and CAMSAP3 CC+CKK domains. For both CAMSAPs, the CC+CKK domains recapitulate minus-end capping to the same extent as the FL proteins. (Scale bars: horizontal, 1 μm; vertical, 20 s.) (D) Quantification of minus-end growth rates determined by long-term (20 min) imaging of MT dynamics (control growth rate from Fig. 2, for CAMSAP2 and -3 CC+CKK, n = 2, n = 14–32 MTs per day). Mean and SEM of each experimental day are shown. Only MTs with GFP initially at the minus-end were analyzed.

Fig. 4.

CAMSAPs protect MT minus-ends from depolymerization by MCAK. (A) Schematic of assay. (B) Representative kymographs of control MT depolymerizing from both ends (Left), and MT protected in the presence of CAMSAP3 (Right). In the low-salt conditions needed for MCAK depolymerization, CAMSAPs tend to bind along MT length; hence, only the MT imaging channel is shown. (Scale bars: horizontal, 1 μm; vertical, 60 s.) (C) Quantification of MTs exhibiting kinesin-13-induced end-wise depolymerization behaviors. Number of independent experiments on separate days are as follows: control, n = 3; CAMSAP1, n = 2; CAMSAP2, n = 2; CAMSAP3, n = 2, for n = 17–142 MTs per day. Mean and SEM are shown for the different experimental days.

Fig. 5.

Domains required for Drosophila Patronin minus-end binding, suppression of growth in vitro, and MT stability in S2 cells. (A) Schematic of Patronin domain structure. The Patronin CC domain and CC+CKK domains bind to MT minus-ends (white arrows) in a kinesin gliding assay. (Scale bar: 2 μm.) (B) Representative kymographs of dynamic MTs capped by Patronin GFP-CC and GFP-CC+CKK. (Scale bars: horizontal, 1 μm; vertical, 20 s.) (C) Quantification of minus-end growth rates in the presence of Patronin truncations. Short-term images were scored for Pat CC; long-term images were scored for Patronin CC+CKK. For control, n = 6; Pat CC, n = 1; Pat CC+CKK, n = 2; n = 10–39 MTs per day; mean and SD shown for each experimental day. (Inset) MT growth (red) from stabilized seeds (blue) after 15 min in presence of Pat CC+CKK (green). (Scale bar: 1 μm.) (D) Quantification of Drosophila rescue experiments. Experiment was performed in duplicate (n = 10–12 cells scored on each day); results from each day are shown as separate bars. (E) Representative cells from rescue experiments. (Insets) Zoom of cell edge; yellow arrows mark representative MT fragments used for scoring. (Scale bars: full cell, 10 μm; Inset, 2.5 μm.)