antibody (1:10,000). The size standards used were ferritin (440 kD), aldolase (158 kD), and BSA (67 kD). Actin assembly kinetics Actin ﬁlament assembly assays were performed in 60-μl reactions as previ- ously described (Moseley et al., 2006). In brief, gel-ﬁltered monomeric actin (ﬁnal concentration 2 μM, 5% pyrene labeled) was converted to Mg- ATP-actin, mixed with the indicated proteins in HKCL buffer or HKCL buffer alone, and then added immediately to 3 μl of 20× initiation mix (40 mM MgCl2, 10 mM ATP, and 1 M KCl). Pyrene ﬂuorescence was monitored at excitation 365 nm and emission 407 nm in a ﬂuorescence spectrophotom- eter (Photon Technology International) held at the constant temperature of 25°C. The rates of actin assembly were determined from the slopes of the assembly curves at 50% polymerization. Epiﬂuorescence and TIRF microscopy Cells were either ﬁxed in 20°C methanol for 10 min and rehydrated in TBS or ﬁxed in 4% PFA in PBS for 10 min followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min. Glu MTs were detected with a polyclonal rabbit antibody SG that speciﬁcally recognizes detyrosinated (Glu) tubulin (Gundersen et al., 1984; 1:400), and tyrosinated (Tyr) MTs were detected with the rat mAb YL1/2 (1:10; European Collection of Ani- mal Cell Cultures). Rhodamine-phalloidin (1:500; Invitrogen) or mouse mAb to actin (C4D6; 1:200) were used to detect the actin cytoskeleton as speciﬁed. Secondary antibodies were obtained from Jackson Immuno- Research Laboratories and were absorbed to minimize cross reaction with other species. Immunostained samples were observed with a microscope (Optiphot; Nikon) using a 60× plan apo objective (1.4 NA) and ﬁlter cubes optimized for coumarin, ﬂuorescein/GFP, rhodamine, and Cy5 ﬂuo- rescence (Nikon). Images were captured with a cooled charge-coupled device camera (MicroMax; KAF 1400 chip; Kodak) using Metamorph soft- ware (MDS Analytical Technologies). For live-cell imaging experiments by TIRF microscopy, preparations were observed using a 60× plan apo objective (1.45 NA) on a micro- scope (TE2000-U; Nikon) equipped with a TIRF illuminator and ﬁber optic– coupled laser illumination. TIRF illumination was obtained with a separate laser line (Ar ion; 488 nm) using a ﬁlter cube optimized for ﬂuorescein/ GFP ﬂuorescence (Chroma Technology Corp.). Images were captured with a cooled charge-coupled device camera (Orca ERI; Hamamatsu) using Metamorph software. For live-cell imaging of GFP-fusion mDia2 constructs by TIRF, cells were transfected 24 h before microscopy and images were acquired every 200 ms in GFP recording medium (Ho et al., 1998) plus 1 mM sodium pyruvate and 1% calf serum at 37°C. Kymographs were generated using Metamorph software from image stacks (47 frames) of the selected regions from each transfected cell as indicated. To measure the speed of each particle, a line region was drawn onto the kymograph and width and length logged into Excel (Microsoft). The mean speed was cal- culated for at least 10 particles. All images for ﬁgures were processed with Photoshop (Adobe). MT binding assays PC or DEAE tubulin at 25 μM (puriﬁed as described in Mikhailov and Gundersen [1995] and Tian et al. [1997]) was polymerized in PEMG buffer (100 mM Pipes-KOH, pH 6.9, 1 mM EGTA, 2 mM MgCl2, and 1 mM GTP) with 10 μM Taxol at 37°C for 1 h. 12.5 μM of polymerized MTs was incu- bated with 1 μM of puriﬁed mDia2 fragments or BSA for 10 min at 30°C. MT pellets were recovered by centrifugation at 100,000 g for 10 min at 37°C, and matching aliquots of input and MT pellets were loaded onto a 10% SDS-PAGE. Proteins were detected by Coomassie staining. Saturation binding was measured using a range of concentrations of His-FH1FH2mDia2 (0–15 μM) and a ﬁxed amount of MTs (1.5 or 3 μM). MT-bound mDia2 was estimated by Coomassie staining by comparison with a protein standard of known concentration run on the same gel. The fraction of mDia2 sediment- ing in the absence of MTs was subtracted from each data point. KD and stoi- chiometry of binding at theoretical saturation were calculated as best ﬁt values using Prism 4 (GraphPad) assuming one site of binding for mDia2 on the MT polymer. MT shearing was obtained by passing the MTs through a 26G needle at least 10 times and was conﬁrmed by immunoﬂuorescence. Immunostaining analyses of His-FH1FH2mDia2 were performed in ﬂow chambers in which 2 μM of rhodamine-labeled and taxol-stabilized MTs were immobilized onto coverslips precoated with mutant rigor kinesin (E164A; provided by S. Gilbert, University of Pittsburgh, Pittsburgh, PA) in the presence of 1 mM ATP. Non-speciﬁc protein binding to glass surfaces was blocked by 1 mg/ml casein before mDia2 perfusion. 2 μM FH1FH2mDia2 in PEM buffer plus additives (10 μM taxol with an oxygen-scavenging cocktail to prevent photodamage, including 220 μg/ml glucose oxidase, 534 JCB • VOLUME 181 • NUMBER 3 • 2008 22.5 mM glucose, 36 μg/ml catalase, and 71.5 mM -mercaptoethanol) or PEM buffer plus additives alone were then perfused in each chamber and incubated with MTs for 5 min. MTs were then washed three times in PEM buffer plus additives, ﬁxed in 1% glutaraldehyde, and processed for immunoﬂuorescence using mouse anti-His and Cy2 anti–mouse for the de- tection of FH1FH2mDia2. Preparations were observed using a 60× plan apo objective (1.45 NA) on a TE2000-U microscope. Images were cap- tured with a cooled charge-coupled device camera (Orca ERII) using Meta- morph software and ﬁgures were assembled with Photoshop. MT stability assays PC or DEAE tubulin at 25 μM was induced to polymerize in PEMG buffer with 10% DMSO. Taxol seeds were used in experiments in which the popu- lation of surviving MTs was assessed by immunostaining. Polymerization re- actions were performed in the presence of 2 μM mDia2 proteins at 37°C for 1 h. To assess MT stability to cold-induced depolymerization, matching aliquots of the samples were centrifuged, as described in the previous sec- tion, to isolate the MT pellet. The remaining sample was then incubated at 4°C for 30 min and spun in an ultracentrifuge (TLA100; Beckman Coulter) for 10 min at 60k rpm at 4°C to isolate cold-stable MTs. Samples of the in- put material, pellets, and supernatants were resolved on 10% SDS-PAGE and the proteins were detected by Coomassie staining. To assess MTs stabil- ity to dilution-induced depolymerization, 25 μM DEAE tubulin was polymer- ized in the presence of taxol seeds to generate longer MTs and recombinant mDia2 fragments at 2 μM were added to MTs diluted 10 times their original volume in PEM buffer (100 mM Pipes-KOH, pH 6.9, 1 mM EGTA, and 2 mM MgCl2) for 10 min at 37°C to induce depolymerization. The reactions were stopped by the addition of 0.5% glutaraldehyde. Matching aliquots of input, diluted, and cold-treated material were then spotted onto poly- lysine–coated coverslips and ﬁxed by incubating with ice-cold methanol for 5 min. MTs were visualized by staining with a rat anti-Tyr tubulin antibody and a secondary Cy3-conjugated anti–rat antibody and epiﬂuorescence microscopy using a microscope (Optiphot) with a 60× plan apo objective (1.4 NA). Images were captured with a cooled charge-coupled device cam- era (MicroMax) using Metamorph software and ﬁgures were assembled using Photoshop. MT bundling before and after dilution was assessed by polymerizing 25 μM MTs using MT seeds in the presence or absence of 2 μM FH1FH2mDia2. MT bundles were sedimented by low-speed centrifugation at 4,500 g for 5 min at room temperature. Matching aliquots of the input materials, pellets, and supernatants were resolved on 10% SDS-PAGE and the proteins were detected by Coomassie staining. MT dynamics analyses MT dynamics analyses were performed with MTs nucleated from sea urchin ﬂagellar axoneme fragments and visualized by video-enhanced DIC microscopy as previously described (Walker et al., 1991; Pryer et al., 1992; Howell et al., 1999). In brief, coverslips were mounted on glass slides using double-stick tape as spacers to create a ﬂow chamber of 10-μl vol. Sea urchin axonemes were allowed to adhere to the coverslip for 5 min. Chambers were then perfused with 50 μl PEM to remove unbound axonemes and then 5 mg/ml casein to block nonspeciﬁc protein binding to glass surfaces. The chamber was then perfused with 50 μl PEM to wash out the casein and ﬁnally perfused with a solution of 10 μM tubulin and 1 mM GTP in PEM. In MT assembly reactions to measure MT dynamics, 2 μM His-FH1FH2mDia2 or buffer alone (HKCL + PEM buffer) was added to the MT assembly mixture. The chamber was then warmed on the microscope stage to 35°C to initiate MT assembly. In most MT disassembly experi- ments, a single microscope ﬁeld was viewed for 2–5 min during assembly and then perfused with either buffer (PEM + HKCL) or solutions containing mDia2 (0.5–4 μM in HKCL). MT growth rates during assembly or before perfusion in MT disassembly experiments were used to deﬁne plus and mi- nus ends of the axonemes. Mean rates of elongation and shortening for individual MTs were determined by least squares regression analysis of changes in MT length as a function of time. Frequencies of catastrophe were determined by dividing the total number of catastrophes by the sum of the total time spent in elongation for all MTs of one polarity. Taxol-stabilized seeds Taxol-stabilized MT seeds were generated by polymerizing DEAE tubulin at 37°C for 30 min with 100 μM GTP and stepwise addition of Taxol (1–20 μM). MTs were centrifuged at 100,000 g for 10 min at 37°C and resuspended in PEM with 100 μM GTP and 20 μM Taxol. Seeds were gen- erated by passing isolated MTs through a 22-gauge syringe 20 times and then isolated by centrifuging at 100,000 g for 20 min at 4°C. Pellets were washed twice with PEM and resuspended in half of their initial volume in PEM with 100 μM GTP.