Research Article 817 1C, a soluble form of syntaxin, attenuates membrane recycling by destabilizing microtubules

Takahiro Nakayama1,*, Hiroyuki Kamiguchi2 and Kimio Akagawa1 1Department of Cell Physiology, Kyorin University School of Medicine, Tokyo, 181-8611, Japan 2Laboratory for Neuronal Growth Mechanisms, RIKEN Brain Science Institute, Saitama, 351-0198, Japan *Author for correspondence ([email protected])

Accepted 20 September 2011 Journal of Cell Science 125, 817–830 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.081943

Summary Syntaxin 1C (STX1C), produced by alternative splicing of the stx1A , is a soluble syntaxin lacking a SNARE domain and a transmembrane domain. It is unclear how soluble syntaxin can control intracellular membrane trafficking. We found that STX1C affected microtubule (MT) dynamics through its tubulin-binding domain (TBD) and regulated recycling of intracellular vesicles carrying glucose transporter-1 (GLUT1). We demonstrated that the amino acid sequence VRSK of the TBD was important for the interaction between STX1C and tubulin and that wild-type STX1C (STX1C-WT), but not the TBD mutant, reduced the Vmax of glucose transport and GLUT1 translocation to the plasma membrane in FRSK cells. Moreover, by time-lapse analysis, we revealed that STX1C-WT suppressed MT stability and vesicle-transport motility in cells expressing GFP–a-tubulin, whereas TBD mutants had no effect. We also identified that GLUT1 was recycled in the 45 minutes after endocytosis and that GLUT1 vesicles moved along with MTs. Finally, we showed, by a recycling assay and FCM analysis, that STX1C-WT delayed the recycling phase of GLUT1 to PM, without affecting the endocytotic process of GLUT1. These data indicate that STX1C delays the GLUT1 recycling phase by suppressing MT stability and vesicle-transport motility through its TBD, providing the first insight into how soluble syntaxin controls membrane trafficking.

Key words: Glucose transporter, Microtubule stability, Recycling, Syntaxin, Vesicle motility

Introduction et al., 2004). STX1C possesses a large part of the N-terminal Soluble N-ethylmaleimide-sensitive factor attachment domain of STX1A, whereas the novel C-terminal domain of 34

Journal of Cell Science receptor (SNARE) machinery is a central regulator of specific residues is generated by the insertion of a 91-bp splicing region. interactions between vesicular v-SNARE (e.g. VAMP; also This insertion results in a loss of the TMD and the functional known as ) and target t-SNARE [e.g. SNAP25 SNARE domain, the helical structure necessary for association and syntaxin (STX)] (Rothman, 1994). The STX family consists with v-SNARE, resulting in cytoplasmic expression (Jagadish of 19 members, most of which associate with specific membrane et al., 1997; Jahn and Sudhof, 1999; Nakayama et al., 2003). It is compartments by virtue of a hydrophobic trans-membrane generally believed that membrane-bound , such as domain (TMD) in their C-terminal region and regulate STX1A, regulate membrane trafficking through the SNARE membrane transport between intracellular compartments and domain (Jahn and Sudhof, 1999). However, the mechanism by target organelles (Teng et al., 2001). For example, STX1A is which the soluble syntaxin STX1C affects GLUT1 translocation involved in the docking and/or fusion of synaptic vesicles to the remains unknown. plasma membrane (PM) at active zones in neurons (Bennett et al., GLUT1, which is a representative member of the high-affinity 1992; Inoue et al., 1992). Several syntaxins have soluble variants facilitative glucose transporter (GLUT) family including lacking a TMD (e.g. STX1BDTMD, STX1C, 2D, 3D, 16C), GLUT3 and GLUT4, is responsible for the entry of glucose which are generated by alternative splicing (Ibaraki et al., 1995; into cells and for maintaining cell metabolism and homeostasis Jagadish et al., 1997; Pereira et al., 2008; Quinones et al., throughout the periphery and the brain (Olson and Pessin, 1996). 1999; Simonsen et al., 1998). However, the functions and the Recently, it has been demonstrated that GLUT translocation to mechanism of action of these soluble syntaxins have not yet been the PM is dependent on the cytoskeletal systems including determined. microtubules (MTs) and actin filaments (Dransfeld et al., To better understand the action of soluble syntaxins, we focused 2001; Singh et al., 1998; Tong et al., 2001; Wang et al., on STX1C. STX1C, an alternative splice variant of the stx1A gene, 1998). For example, drugs that influence the function of tubulin is deleted hemizygously in patients with the neurodevelopmental polymerization, such as vinblastine and paclitaxel, alter glucose disorder Williams syndrome, which shows characteristic cognitive uptake activity by GLUT1 in glioma cells (Singh et al., 1998). It profiles, such as hyperactivity, poor attention span, good memory, has also been reported that the GLUT4 vesicular transport remarkably spared linguistic abilities and visual spatial deficits depends on cortical actin remodeling (Dransfeld et al., 2001; (Nakayama et al., 1997; Nakayama et al., 1998). STX1C inhibits Tong et al., 2001; Wang et al., 1998) that is regulated by a direct intracellular glucose transport by suppressing the translocation of link between SNARE and the MT network (Pooley glucose transporter-1 (GLUT1) to the PM in glial cells (Nakayama et al., 2008). Furthermore, MT disruption by nocodazole inhibits 818 Journal of Cell Science 125 (4)

the motility of the vesicle-transport (VT)-containing PM protein did not know whether exogenous STX1C and the TBD affected reduced folate carrier (RFC), as well as final translocation of glucose uptake activity or GLUT1 translocation in epithelial RFC to the PM in epithelial cells (Marchant et al., 2002). cells. Therefore, to understand the effect of STX1C and the TBD Interestingly, the STX1C N-terminal region possesses a tubulin- on glucose uptake activity, we exogenously introduced DsRed binding domain (TBD) that is found in the MAP2 and tau vector (Vec), STX1C-WT, STX1C-AAAA or STX1C-GGGG to protein family (Lewis et al., 1988) (hereafter referred to as at least three different transformant cell lines for each construct. MAP2/tau). In MAP2/tau, the TBD regulates MT dynamics Western blotting analysis with the 14D8 antibody, which is together with regulatory proteins, including MT plus-end- reactive to common N-termini of STX1C and STX1A, showed binding proteins and soluble factors, such as cytoplasmic that the expression of each introduced construct in the various linker proteins (CLIPs) and stathmin (Howard and Hyman, transformants was almost the same (Fig. 1C). We also found that 2009). This leads us to postulate that the TBD of STX1C also GLUT1 was the most abundant of the high affinity glucose affects cytoskeletal transport systems. transporters in FRSK cells and that the amount of GLUT1 protein In this study we used an lung epithelial cell line, FRSK, to was similar among the transformants (Fig. 1C), indicating that examine the function of the N-terminal TBD of STX1C overexpression of the exogenous STX1C constructs did not affect to elucidate how this soluble syntaxin suppresses GLUT1 GLUT1 expression in FRSK cells. translocation. Our data indicate that STX1C delays the Next, we investigated glucose uptake by GLUT1 using 2- recycling phase of GLUT1 to the PM. Thus, we report the first deoxy-D-glucose (2-DG), a non-metabolic glucose analog. As characterization of the activity of a soluble syntaxin and establish shown in Fig. 1D a 2-DG kinetic assay was carried out using 0.1– that STX1C alters the cellular distribution of GLUT1 through 100 mM 2-DG, to examine the Michaelis constant (Km) and the suppression of MT stability and VT motility through its TBD. maximum velocity of the reaction (Vmax). The Vmax value was This suggests a functional role of STX1C in the suppression of significantly reduced in the transformant cells expressing GLUT1 translocation and intracellular glucose transport. STX1C-WT without changing the Km value, compared with the FRSK parent cells (Pt), cells expressing Vec and TBD-mutated Results constructs (AAAA, GGGG; Table 1). This result was also STX1C suppresses glucose transporter-1 (GLUT1) confirmed by the Eadie–Hofstee plot (Fig. 1D, inset). This translocation and glucose transport activity through the reduced Vmax value was not caused by a change in basal glucose tubulin-binding domain transport by the sodium-dependent glucose transporter (SGLT), To investigate how the TBD of STX1C is involved in MT because 2-DG uptake through SGLT, measured under sodium- polymerization in vivo, we first examined the association free conditions, accounted for only ,10% of the total 2-DG between STX1C and tubulin by using wild-type (WT) and uptake and was not altered by overexpression of exogenous mutated peptides of the predicted TBD that correspond to constructs in epithelial cells (data not shown) or glioblastoma residues 89–106 of STX1C. As shown in Fig. 1A, given that half cells, as reported previously (Nakayama et al., 2004). These of the residues (i.e. 89–98) of STX1C show high homology with results suggest that the suppression of glucose uptake by STX1C- the TBD of MAP2/tau, we attempted to replace the first four WT might be due to a decrease in the number of GLUT1

Journal of Cell Science residues of the peptide; valine, arginine, serine and lysine molecules on the PM, but not to a change in the glucose transport (VRSK), with alanines (AAAA) or glycines (GGGG). In rate by a single GLUT1 molecule. competition assays the WT peptide significantly inhibited the Finally, to interpret results of the kinetic assay, we examined association between recombinant His-tagged STX1C and tubulin the effect of STX1C TBD on GLUT1 translocation in each of the at 0.5 and 5 mM compared with 0 mM of WT competitor, transformant cells. Using immunofluorescence microscopy, we whereas the mutated peptides (AAAA and GGGG) did not out- found that exogenous STX1C-WT was expressed in the cytosol, compete the association between recombinant His-tagged STX1C and suppressed GLUT1 translocation to the PM (Fig. 1E). and tubulin (Fig. 1B). This indicates that the VRSK sequence in Furthermore, to investigate the effect of STX1C TBD mutants TBD is crucial for the interaction between STX1C and tubulin. on GLUT1 translocation, mutated constructs were transfected Thus, we prepared several DsRed–STX1C expression into FRSK cells. In contrast to STX1C-WT, expression of mock constructs with WT or mutated TBDs (pDsRed–STX1C-WT, vector or TBD-mutated constructs (STX1C-AAAA and -GGGG), and STX1C-AAAA and STX1C-GGGG, respectively) for did not suppress GLUT1 translocation (Fig. 1E). Similar results transfection experiments. Next, for the in vivo MT- were also obtained in cells transiently transfected with each polymerization assay, we selected several types of cell that did construct (data not shown). These results suggest that the TBD not endogenously express STX1C or STX1A, both of which are domain of STX1C is also responsible for the suppression GLUT1 alternative splice variants of the stx1A gene with a common translocation. N-terminus. We did this in order to avoid the potentially complicating effects on dynamics caused by endogenous wild- STX1C suppresses the dynamicity of pioneer MTs through type STX1C isoforms, and to more directly identify loss of the TBD function on dynamics by the STX1C TBD mutants as shown in a To investigate the mechanism of suppression, cloned FRSK cells previous study on the effect of Tau mutants on dynamics (Bunker stably expressing GFP–a-tubulin were transfected with STX1C et al., 2006). We cloned cell lines stably expressing GFP–a- constructs and the dynamics of MT polymerization was tubulin and chose a lung epithelial cell line, FRSK, which has visualized using time-lapse microscopy. Fig. 2A shows a thin cell periphery suitable for in vivo imaging (Fig. 1C). representative fluorescence time-lapse image of living FRSK We previously reported that STX1C suppresses glucose cells stably expressing GFP–a-tubulin. As shown in Fig. 2B, we transport through the inhibition of GLUT1 translocation to the captured time-lapse images of pioneer MTs in the flat peripheral PM in glioblastoma cells (Nakayama et al., 2004). However, we region of these cells. Most MT plus-ends were oriented toward STX1C regulates the GLUT1 recycling phase 819 Journal of Cell Science

Fig. 1. Effect of the STX1C TBD on glucose transport activity and GLUT1 translocation. (A) The tubulin-binding domain (TBD) in the N-terminus of STX1C. Tubulin-binding sequences of known molecules and STX1C are aligned. Identical and homologous amino acids are shaded orange and yellow, respectively. STX1C-AAAA and STX1C-GGGG are constructs mutated in the ‘VRSK’ site of the tubulin-binding motif of STX1C-WT. (B) Competitive in vitro binding assay. Precipitation by His-STX1C in conditions containing tubulin fractions and 0–5 mM peptide competitors (WT, AAAA or GGGG) were immunoblotted with tubulin or STX1C antibodies. Quantified results from the blotting are shown below. Statistical significance was determined using a t-test with equal variance; *P,0.05 and **P,0.01 versus 0 mM competitor. (C) Immunoblot of the high-affinity GLUT family members in FRSK transformants. Transformant cells were cloned from cells transfected with DsRed mock vector (Vec), STX1C-WT (WT), STX1C-AAAA (AAAA) and STX1C-GGGG (GGGG) constructs. Lysate from each FRSK transformant was immunoblotted with antibodies against STX1C and high-affinity GLUT (GLUT1, -3, -4). (D) Kinetic analysis of 2-DG uptake in FRSK parent (Pt) and transformant cells stably expressing exogenous constructs. The dose-dependent analysis was carried out as described in the Materials and Methods. Values were obtained from three independent experiments. Pt (R250.999), Vec (R250.999), WT (R250.999), AAAA (R250.999), GGGG (R250.998). An Eadie–Hofstee plot is shown in the inset. (E) Immunofluorescence of GLUT1 in FRSK cells stably expressing exogenous constructs. The left panel (a,d,g,i; green) includes confocal images observed using the GLUT1 antibody. The middle panel (b,e,h,k; red) includes confocal images observed using the STX1C antibody. The right panel (c,f,i,l) shows merged images. 820 Journal of Cell Science 125 (4)

Table 1. Effect of STX1C TBD on the uptake of 2-deoxy-glucose (2-DG) Pt Vec WT AAAA GGGG ,{,{ Vmax (nmol/min/mg) 8.561.1 7.161.7 4.260.4* 6.660.4 5.960.4 Km (mM) 28.967.3 27.267.7 26.261.4 27.462.2 32.461.1

Values of Km and Vmax obtained from 2-DG kinetic assay were compared between parent cells (Pt) and each transformant with DsRed mock vector (Vec), STX1C-WT (WT), STX1C-AAAA (AAAA) or STX1C-GGGG (GGGG). Values are means 6 s.e.m. from three independent experiments. Statistical significance was determined using a t-test with equal variance. *P,0.05 vs Vec; {P,0.05 vs AAAA; {P,0.05 vs Pt (parent cells). Journal of Cell Science

Fig. 2. MT polymerization in FRSK cells expressing GFP–a-tubulin. (A) Whole- mount view of a representative cloned cell expressing GFP–a-tubulin, observed by fluorescence microscopy: FRSK cells transfected with the GFP–a-tubulin construct were selected using G418 and cloned to produce as a single cell line. (B) Time-lapse sequences of MTs in untreated live FRSK cell stably expressing GFP–a-tubulin: the white rectangle indicates the region used for the time-sequence images shown below. Arrowheads indicate the tip of the same MT. The frame numbers are shown in the bottom left corner. The duration of the sequence shown was 2.155 seconds. The dynamic phases of the MTs is shown across the bottom. (C) Life-history plots of the dynamics of individual MTs. The positions of the ends of the individual MT were tracked in cells transfected with exogenous constructs. Changes in the length of the MTs were plotted versus time. Each line represents a single MT. STX1C regulates the GLUT1 recycling phase 821

the leading edge and exhibit non-equilibrium polymerization behavior, referred to as dynamic instability, with stochastic switching between phases of growth and shortening, consistent with previous studies in endothelial and fibroblast cells (Desai and Mitchison, 1997; Wittmann and Waterman-Storer, 2001). Fig. 2B also shows typical dynamic MT transition between the three phases (i.e. growth, shortening and attenuation) as defined in the Materials and Methods (see also supplementary material Movie 1). Typical MT life-history plots for cells transfected with mock vector (Vec), STX1C-WT, STX1C-AAAA or STX1C- GGGG are presented in Fig. 2C (see also supplementary material Movies 2–5). From these individual MTs, we determined the dynamic instability of MTs, as described in the Materials and Methods. We first assessed the ability of each construct to influence the velocity and distance that dynamic MTs exhibit in the growth or shortening phase. As shown in Fig. 3A, STX1C-WT only significantly (P,0.01) reduced the velocity of MT in the shortening phase, whereas the velocity during the growth phase remained unchanged relative to MTs in Vec, STX1C-AAAA or STX1C-GGGG. By contrast, the ability to influence MT dynamics was not significantly different between the four constructs (Fig. 3B). STX1C-WT increased the attenuation time of MTs, compared with Vec, STX1C-AAAA or STX1C-GGGG; the two TBD mutants (AAAA and GGGG) having no effect (Fig. 3C). We then calculated the fraction of time that dynamic MTs spent in each phase relative to the total time tracked. We found that STX1C-WT significantly (P,0.01) influenced the fraction of time that dynamic MTs spent in each phase relative to Vec, STX1C-AAAA and STX1C-GGGG (Table 2). For example, STX1C-WT significantly (P,0.01) increased the fraction of time MTs spent attenuated, by 25.3% (from 44.4 to 59.4%; Table 2), while reducing the fraction of time MTs spent growing and

Journal of Cell Science shortening by 42.3% (from 30.1 to 17.3%; Table 2) and 8.6% (from 25.5 to 23.3%; Table 2) respectively, relative to the Vec treatment (Table 2). By contrast, the two STX1C TBD mutants did not influence the phase distribution of the MTs relative to that of the Vec treatment. To determine whether the different STX1C constructs affected MT dynamicity, the percentage of dynamic MTs versus total MTs in the cells was initially analyzed using the criteria described in the Materials and Methods. For the analysis of the Fig. 3. Effect of the STX1C TBD on MT polymerization in pioneer MTs. density of total MTs per analyzed area, a region of the cell (A,B) Effect of exogenous STX1C on velocity and distance of MT periphery was randomly chosen, and the number of MTs in the polymerization in pioneer MTs. The MT dynamic velocity (A) and distance area was counted. STX1C-WT significantly (P,0.01) decreased (B) were determined from MTs in flat peripheral regions of the parent cell the density of total MTs relative to MTs in Vec- or STX1C- (Pt) or cells transfected with exogenous constructs. The total number of AAAA-treated cells and the density of dynamic MTs relative to shortening and growth events observed from at least 38 MTs and 12 cells is: MTs in Vec-, STX1C-AAAA- and STX1C-GGGG-treated cells Pt, 195 and 190; Vec, 185 and 171; WT, 198 and 170; AAAA, 187 and 166; (Table 3). Additionally, STX1C-WT significantly (P,0.01) and GGGG, 199 and 183, respectively. Statistical significance was decreased the percentage of dynamic MTs versus total-MTs, by determined using a one-way ANOVA and multiple-comparison test; 24% (from 86.6 to 65.8%; Table 3) relative to MTs in Vec- **P,0.01 versus Pt, Vec, AAAA and GGGG. (C) Effect of exogenous treated cells (Table 3). By contrast, two of the STX1C TBD STX1C on pause time of MT polymerization in pioneer MTs. The total event number analyzed is: Pt, 140; Vec, 100; WT, 100; AAAA, 100; and GGGG, mutants did not influence the density of dynamic or total MTs or 100. Statistical significance was determined using a one-way ANOVA and a the percentage of dynamic MT relative to MTs in Vec-treated multiple-comparison test; **P,0.01 versus Pt, Vec, AAAA and GGGG. cells. In particular, the STX1C-AAAA mutant was significantly (P,0.01) compromised in all abilities to influence the density of dynamic or total-MTs and the percentage of dynamic MT, population. It was calculated as the total length grown and compared with STX1C-WT. A similar result was obtained from shortened divided by the time period observed through the the analysis of the dynamicity, which is a measure of the visually process of dynamic instability. STX1C-WT decreased the detectable amount of dynamic instability occurring in a MT dynamicity of MTs compared with those in the Vec control 822 Journal of Cell Science 125 (4)

Table 2. Effect of STX1C TBD on the fraction of time S2). Next, according to an established method for examining microtubules spent growing, shortening and pausing membrane recycling (Kamiguchi et al., 1998), we investigated the fate of endocytosed GLUT1 using the GLUT1-ectodomain Fraction of time spent (%) antibody (see the Materials and Methods). Fig. 4A illustrates that Growing Pausing Shortening internalized-GLUT1 molecules were recycled back to PM after Pt 27.862.2 49.663.0 22.561.6 an extra 45 minutes in culture (Fig. 4Ag,h,i), whereas at time- Vec 30.162.1 44.462.3 25.561.5 ,{,{,§ ,{,{,§ points excepting 45 minutes, recycling of GLUT1 molecules was WT 17.363.0* 59.465.5* 23.363.2 not detected (Fig. 4A). AAAA 28.563.7 43.765.6 27.862.7 GGGG 26.463.0 47.264.8 26.462.3 To investigate VT motility in FRSK cells, we used the fluorescent probe FM1-43, which is useful for measuring Fraction of time spent was compared between parent cells (Pt) and cells membrane trafficking (Brumback et al., 2004). Using this transfected with exogenous DsRed constructs. Statistical significance was fluorescent probe, we first studied the colocalization of determined using one-way ANOVA and multiple-comparison test as follows: *P,0.01 vs Pt; {P,0.01 vs Vec; {P,0.01 vs AAAA; §P,0.01 vs FM1-43 vesicles and GLUT1 vesicles in FRSK cells. Double GGGG. immunostaining using anti-GLUT1 IgG and FM1-43FX, a fixable analog of FM1-43, revealed that FRSK cells have a heterogeneous population of single or aggregated vesicles of cells (from 21 to 18.8%, a decrease of 11%; Table 3), whereas ,0.5-3 mm diameter. This is consistent with the observed vesicle the two TBD mutants had no effect (Table 3). size in a variety of epithelial cells reported previously (Jerdeva We also tried to perform time-lapse analysis by using et al., 2005; Sahlen et al., 2002). Double immunostaining U87MG glioblastoma cells expressing endogenous STX1C revealed that 95.0460.71% (33 cells) of FM1-43FX vesicles (supplementary material Fig. S1). The effect of STX1C TBD that colocalized with GLUT1 vesicles in the flat peripheral region mutants on MT polymerization indexes in cells expressing were #1.5 mm diameter (Fig. 4B). Therefore, FM1-43 vesicles endogenous STX1C was similar to those in cells not expressing #1.5 mm in diameter in the FRSK cell periphery could be used to endogenous STX1C (supplementary material Table S1), except monitor GLUT1 vesicles. We also examined the colocalization for the effect on the velocity and distance in the growth phase, of FM1-43FX vesicles and MTs in FRSK cells. Double which might be due to other factors in cells expressing immunostaining using FM1-43FX and anti-b-tubulin IgG endogenous STX1C. The result suggests that the effect on MT revealed that 99.5260.10% (86 cells) of vesicles #1.5 mm polymerization indexes in FRSK cells not expressing endogenous diameter in the flat peripheral region colocalized with MTs STX1C is not the result of side-effects of other factors. (Fig. 4C). To assess whether vesicles in the flat peripheral region Thus, these data suggest that, because a similar effect was also moved with MTs, we further studied VT motility in living cells obtained in U87MG-expressing endogenous STX1C, STX1C- stably expressing GFP–a-tubulin using FM4-64, an alternative WT plays a role as a negative regulator, leading to the analog of FM1-43. Simultaneous time-lapse observations with suppression of MT stability through its TBD. GFP and FM4-64 using a DualView system revealed that vesicles in the flat peripheral region were actually moving along with Journal of Cell Science STX1C suppresses GLUT1-vesicle transport through its MTs (Fig. 4D). These data indicate that GLUT1 vesicles #1.5 TBD mm in diameter, move with MTs. MT dynamics are related to the motility of vesicles containing On the basis of these observations, we performed live PM protein (Marchant et al., 2002). Thus, we attempted to study microscopy studies on vesicles moving with MTs in living the effect of STX1C on VT motility. It is generally accepted that cells. Fig. 5A shows a representative FM1-43 time-lapse image GLUT1 is expressed and recycled constitutively (Olson and of a living parent FRSK cell. The FM1-43 fluorescent signal Pessin, 1996), however, the recycling process remains unclear. was captured as time-sequential images (Fig. 5A, lower Thus, we investigated GLUT1 recycling using an anti-GLUT1 panel). Vesicles, visualized as punctate signals showed monoclonal IgG. Given that this antibody detects the ectodomain complex transport behavior, which included long-range bi- of GLUT1, depending on the kind of cell (Kinet et al., 2007; directional motions and short-range motions, such as rotation Mueckler et al., 1985), we first confirmed that the monoclonal and oscillation, in FRSK cells (see also supplementary material antibody was useful as an antibody recognizing the extracellular Movie 6). To assess the exact motility of secretory vesicles domain of GLUT1 in FRSK cells (supplementary material Fig. moving with MTs, with the exception of vesicles rotating and

Table 3. Effect of STX1C-TBD on dynamicity of pioneer microtubules Total MT Dynamic MT Dynamic MT Dynamicity No. of cells (No./mm2) (No./mm2) (%) (mm/minute) Pt 33 0.090260.0061 0.077060.0045 86.262.1 21.7 Vec 22 0.095560.0073 0.082660.0069 86.662.6 21.0 WT 27 0.062060.0044*,{,{ 0.042260.0048*,{,{,§ 65.863.9*,{,{,§ 18.8 AAAA 26 0.098960.0058 0.079560.0045 80.763.5 22.4 GGGG 23 0.083160.0046 0.067160.0046 80.562.7 21.3

Density or rate values of total and dynamic microtubules (MTs) are given as means 6 s.e.m. Statistical significance was determined using one-way ANOVA and multiple-comparison test as follows: *P,0.01 vs Pt; {P,0.01, {{P,0.05 vs Vec; {P,0.01, {{P,0.05 vs AAAA; §P,0.01 vs GGGG. STX1C regulates the GLUT1 recycling phase 823 Journal of Cell Science

Fig. 4. Characterization of MT dependent GLUT1 vesicle transport in FRSK cells. (A) CCD images of immunofluorescence of FRSK cells showing the recycling of GLUT1 during the 15–75 minutes after endocytosis of the GLUT1 antibody for 30 minutes. The left panels (a,d,g,k,n; green) show GLUT1 antibody. The middle panels (b,e,h,l,o) are DIC images. The right panels (c,f,i,m,p) are merged images. (B) Colocalization of FM1–43FX vesicles and GLUT1 vesicles in FRSK cells. Cells were cultured in medium including FM1–43FX, a fixable analog of FM1-43 and a GLUT1 ectodomain antibody. The middle panels (c,i) are merged CCD images of the FM1-43FX signal (green; a,g) and GLUT1 (red; b,h). The right panels (f,k) are merged images of the DIC signal (e,j) and fluorescence (green+red; c,i). The regions in the white rectangles (upper figure) are shown below, at higher magnification. Arrowheads indicate vesicles containing colocalized FM1–43 and the GLUT1. Scale bars: 2 mm. (C) Colocalization of FM1-43FX vesicles and MTs in FRSK cells. The left panels (a,d,g; red) are CCD images of anti-b-tubulin antibody. The middle panels (b,e,h; green) are images of FM1-43FX. The right panels (c,f,i) are merged images. The regions in the white rectangles (1 and 2) in c are shown in d,e,f and g,h,i, respectively. Scale bars: 2 mm. (D) Dual time-lapse sequences of FM4-64 vesicles in living FRSK cells stably expressing GFP–a-tubulin. Arrowheads indicate the same dynamic FM4-64 vesicle on the MT (indicated by an open arrowhead in image 1, and the path is traced with a dotted line in images 5–8). The frame rate was 3.438 seconds/frame. Scale bars: 2 mm.

oscillating, only FM1-43 vesicles anterogradely moving toward motions or disappeared (Materials and Methods). Time-lapse the PM in the flat peripheral region were analyzed until the observations with FM dye also revealed that as the diameter vesicle moved in another direction, switched to short-range increased to .2–3 mm, the vesicles in the FRSK cells gradually 824 Journal of Cell Science 125 (4)

Fig. 5. Effect of the STX1C TBD on the motility of vesicle transport in FRSK cells. (A) Time-lapse sequences of FM1-43 vesicles in a live FRSK parental cell. The white rectangles in the top image show the positions of the sequences in the lower panels. Arrowheads indicate the same dynamic FM1-43 vesicle. The frame numbers are shown in the bottom left corner. The frame rate is 3.038 seconds/frame. (B) Fluorescence micrograph and time-lapse sequences of FM1-43 vesicles in living FRSK cell transfected with exogenous constructs. A fluorescence micrograph of DsRed is shown in the top right corner of each image. For tracking and motility analysis, a region of interest (rectangle) was selected in peripheral regions of the cell. The time sequence images in the rectangle of Vec, AAAA and WT are shown in a, b and c, Journal of Cell Science respectively. Arrowheads indicate the same dynamic FM1-43 vesicle. The numbers on the horizontal and vertical lines are the elapsed time (s) and distance (mm), respectively. Scale bars: 2 mm. (C) Life- history plots of the motility of individual vesicles. Only FM1-43 vesicles moving towards the PM were analyzed. The positions of the ends of individual vesicles were tracked in cells transfected with exogenous constructs. Changes in the distance (upper) and velocity (lower) of the vesicle were plotted versus elapsed time. Each line represents a single vesicle.

lost long-range motion. Thus, only FM1-43 vesicles of ,0.5– second, which is similar to the value of MT-dependent velocity of 1.5 mm in the flat peripheral region were analyzed. To study the vesicles in epithelial and endothelial cells (Manneville et al., effects of STX1C and the TBD on VT motility, cells were 2003; Marchant et al., 2002). A detailed velocity analysis transfected with Vec, STX1C-WT or STX1C-AAAA, and revealed that STX1C-WT significantly (P,0.01) suppressed the changes in movement distance of individual vesicles were average velocity of MT-dependent VT by ,60% compared with measured over time by tracking the positions of directional Vec and STX1C-AAAA (Fig. 5C, upper panel; Table 4), movement ends before another directional movement or whereas there was no difference in the distance of directional disappearance. Fig. 5B,C shows representative FM1-43 time- vesicle movement between constructs (Fig. 5C, lower panel; lapse images and vesicle movement plots, respectively, for cells Table 4). These data indicate that the time parameter in the transfected with a mock vector, STX1C-WT or STX1C-AAAA. equation (velocity5distance/time) is enlarged by STX1C-WT, A VT velocity analysis revealed that epithelial GLUT1 vesicles resulting in slow MT-dependent VT, and suggest that the TBD of were transported with a maximum velocity of ,1.2 mm/second STX1C also affects the transport velocity of MT-dependent and an average velocity during a long-range run of ,0.3 mm/ secretory vesicles. STX1C regulates the GLUT1 recycling phase 825

The TBD of STX1C delays the recycling phase of GLUT1 that the amount of GLUT1 on the PM in first internalization step vesicles had already been suppressed (Fig. 1D–E). To quantify GLUT1 Next, we investigated the effect of STX1C on the recycling in the process of internalization and recycling we conducted profile of GLUT1 after its endocytosis. As in Fig. 4B, GLUT1 flow cytometry (FCM) analysis using the GLUT1 ectodomain recycling periods were examined but with an extra culture period antibody (see the Materials and Methods). Trypsin treatment did of 15–75 minutes after incubation with GLUT1 IgG, and masking not affect the GLUT1 recycling profile in FRSK cells with unconjugated secondary antibody. Fig. 6A–E shows images (supplementary material Fig. S3). Therefore, using FCM at 30, 45, 60, 75 and 90 minutes time points after incubation with analysis in permeabilized cells, we quantitatively evaluated the GLUT1 IgG and extra culture of cells transfected with Vec, endocytotic (Fig. 7A) and recycling (Fig. 7B) processes using STX1C-WT or STX1C-AAAA. At the 45-minute time point, Qdot705-conjugated secondary antibody, because this fluorescent which is the recycling time of GLUT1 to the PM (as shown in analog showed a better signal-to-noise ratio than Alexa Fluor 488 Fig. 4A), positive labeling with Alexa-Fluor-488-conjugated or Alexa Fluor 680R-PE (data not shown). The FCM secondary antibody in cells transfected with Vec, STX1C-WT analysis demonstrated that there was a reduction in the most or STX1C-AAAA was detected in 50.6, 21.0 and 49.4%, frequent signal intensity of Qdot705 bound to the endocytosed respectively, of at least 174 cells examined (Fig. 6B; Table 5). antibody–GLUT1 complex in cells stably expressing STX1C- STX1C-WT suppressed the cell-recycling rate of GLUT1 to the WT, but not those expressing Vec or STX1C-AAAA (Fig. 7A, PM by 58% compared with Vec and STX1C-AAAA. By contrast, lower panel). Furthermore, STX1C-WT decreased the average STX1C-WT increased the rate of cell recycling of GLUT1 to the mean fluorescence intensity (MFI) of Qdot705 bound to PM at the 60 minutes, 75 minutes and 90 minutes compared with endocytosed-GLUT1 by 14% or 15% compared with Vec or Vec, and at the 60 minutes and 90 minutes compared with STX1C-AAAA, respectively (Table 6), as shown by 11 STX1C-AAAA (Fig. 6C–E; Table 5). This is probably the result independent experiments. This result indicates a reduction of of GLUT1 vesicles not arriving at the PM at 45 minutes in internalized GLUT1 molecules, which is consistent with GLUT1 STX1C-WT-treated cells, but instead being delayed and arriving suppression on the PM by STX1C (Fig. 1D–E) and a reduction of endocytosed GLUT1 molecules per one internalized vesicle from at the PM at 60 and 75 minutes. At 15 and 30 minutes, GLUT1 Fig. 6G. Similarly, STX1C-WT reduced the most frequent signal recycling to the PM was only observed at extremely low levels in intensity of Qdot705 bound to GLUT1 that was recycled back to cells with each construct (Fig. 6A; Table 5). These observations the PM (Fig. 7B, lower panel) by 30 or 15% of that seen with demonstrated that STX1C-WT decreased the rate of cell Vec or STX1C-AAAA, respectively, as demonstrated in nine recycling of GLUT1 to the PM at the 45 minutes time point independent experiments (Table 6). Furthermore, the ratio of and increased it thereafter, compared with Vec and STX1C- recycled GLUT1 to endocytosed GLUT1 decreased in cells with AAAA (Fig. 6A–E; Table 5). STX1C-WT, which did not occur with Vec and STX1C-AAAA We further examined whether this effect on the recycling (Table 6). These quantitative observations indicate that the profile was due to an alteration of the GLUT1 internalization reduction of recycled GLUT1 at 45 minutes caused by STX1C- process by STX1C. Fig. 6F shows immunofluorescence images WT reflected a decrease in the quantities of endocytosed GLUT1, of endocytosed GLUT1 using a GLUT1 ectodomain antibody Journal of Cell Science and that STX1C-WT also decreased recycling of GLUT1 at this (see the Materials and Methods). The average density (number time. per mm2) of GLUT1 vesicles in the peripheral region was calculated in at least 81 cells. An internalization assay of GLUT1 Discussion demonstrated that STX1C-WT did not significantly (P.0.05) We demonstrated that STX1C with a wild-type TBD suppressed alter the density of endocytosed GLUT1 vesicles compared with the growth phase instead of increasing the pause phase in MT Vec or STX1C-AAAA (Fig. 6G). polymerization, as well as reducing the MT dynamicity and the Conversely, during microscopic observation, we noted that the density of total and dynamic MTs, suggesting that STX1C acts as signal of GLUT1 recycled to PM at 45 minutes appeared to be a negative factor for MT stability. Additionally, the TBD of weaker in cells transfected with STX1C-WT than the other STX1C was found to be responsible for the suppression of VT constructs. Thus, we next examined whether the observed velocity of the GLUT1 secretory vesicle and delay in the decrease in GLUT1 recycled to the PM by STX1C at this stage recycling phase of the GLUT1 vesicle, but did not affect the was because less GLUT1 was internalized, given the possibility endocytotic process of GLUT1. These observations suggest that suppression of the MT dynamics and VT velocity of GLUT1 Table 4. Effect of STX1C TBD on velocity of vesicle secretory vesicles delays the recycling phase of the GLUT1 transport in the cell peripheral region vesicle, resulting in increased intracellular accumulation of GLUT1 vesicles through alteration of the cellular distribution Velocity Distance of GLUT1. Additionally, these phenomena were not affected by No. of vesicles (mm/second) (mm) the inserted portion unique to STX1C (amino acids 226–260), Pt 83 0.28860.025 3.8060.21 because the TBD mutant with the unique portion (AAAA) did not Vec 46 0.26760.012 3.2360.19 inhibit glucose transport or MT-dynamics. The unique portion WT 37 0.15760.011*,{,{ 2.4960.18* with repetitive proline residues causes loss of the functional AAAA 40 0.25660.015 2.7460.19 helical structure and the TMD, but does not affect MT dynamics. Values of velocity and distance are given as means 6 s.e.m. We demonstrated that STX1C with a wild-type TBD Statistical significance was determined using one-way ANOVA and a associated directly with tubulin and reduced the percentage of multiple-comparison test as follows: { { growth time, decreasing the velocity and the density of total *P,0.01 vs Pt; P,0.01 vs Vec; P,0.01 vs AAAA. and dynamic MTs, the percentage of dynamic MTs and the 826 Journal of Cell Science 125 (4) Journal of Cell Science

Fig. 6. Effect of the STX1C TBD on the process of endocytosis and recycling of GLUT1. (A–E) Immunofluorescence of FRSK cells stably expressing exogenous constructs. GLUT1 endocytosed together with antibody recognizing cell-surface GLUT1 was recycled for 15–90 minutes. The CCD images show immunofluorescence of FRSK cells recycled for 30 minutes (A), 45 minutes (B), 60 minutes (C), 75 minutes (D) or 90 minutes (E) after endocytosis of the GLUT1 antibody for 30 minutes, followed by blocking of IgG on the PM. The left panels (a,d,g; green) are images observed using the GLUT1 antibody. The middle panels (b,e,h; red) are DsRed images. The right panels (c,f,i) are merged images. (F) Immunofluorescence of FRSK cells that endocytosed GLUT1 with antibody recognizing cell-surface GLUT1. Cells were fixed and visualized with Alexa-Fluor-488-conjugated secondary antibody after endocytosis of GLUT1 antibody for 30 minutes, followed by blocking of IgG on the PM. The left panels (a,c,e; green) are images observed using the GLUT1 antibody. The inset figures (a,c,e; red) are images observed using the DsRed signal. The right panels (b,d,f; green) are enlarged images of the boxed regions in the left panels. (G) Effect of exogenous STX1C on the endocytotic process in parent cells (Pt) and cells transfected with exogenous constructs. The total numbers of cells observed with endocytosed GLUT1 vesicles were: Pt, 130; Vec, 103; WT, 81; AAAA, 120. Values are means 6 s.e.m. Statistical significance was determined using a one-way ANOVA and a multiple comparisons test. STX1C regulates the GLUT1 recycling phase 827

Table 5. Effect of STX1C-TBD on ratio of cells recycling GLUT1 to PM % of cell recycling GLUT1 to PM (no. observed) 15 minutes 30 minutes 45 minutes 60 minutes 75 minutes 90 minutes Vec 0.00 (151) 0.46 (217) 50.60 (251) 0.44 (227) 0.00 (182) 0.00 (224) WT 0.00 (174) 0.51 (198) 21.03 (195)*,{ 3.80 (184)**,{{ 3.13 (192)** 9.71 (206)*,{ AAAA 0.00 (185) 0.55 (183) 49.43 (174) 0.51 (195) 0.52 (191) 0.00 (195)

The number of GLUT1-positive cells on the PM was counted in cells transfected with exogenous DsRed constructs. The rate of recycling of GLUT1 to the PM was analyzed in each transfected cell. Statistical significance was determined using Fisher’s test as follows: *P,0.01, **P,0.05 vs Vec; {P,0.01, {{P,0.05 vs AAAA; {P,0.01, {{P,0.05 vs AAAA.

dynamicity, while increasing the time and the percentage of time density, it appears that MAP/tau acts as a MT stabilizing factor spent in the attenuated state of MT polymerization. This (Howard and Hyman, 2009), whereas STX1C acts as a negative is consistent with previous in vitro studies using artificial factor for MT stability. Additionally, STX1C did not result STX1ADTM, which has almost the same N-terminal sequence in binding along the length of MTs, as did MAP/tau, or in as that of STX1C, with the exception of the last half of the accumulation at the plus-end of MTs, as did MT plus-end SNARE domain; artificial STX1ADTM protein associated with tracking proteins (Howard and Hyman, 2009). Additionally, it tubulin and suppressed in vitro MT polymerization activity in a appeared to be cytoplasmic (Nakayama et al., 2003). It has been reassembly assay (Fujiwara et al., 1997; Itoh et al., 1999). These reported that MT density is reduced in cells with increased characteristics appear to be partially compatible with the stathmin, which is an MT-instability factor distributed throughout dynamics of tubulin polymerization in MAP/tau, which showed the cytoplasm, and that it reduces available tubulin subunits by a marked increase in the percentage of time MTs spend in the directly binding to the tubulin subunit, but not to MTs (Jourdain attenuated state as well as the percentage of non-dynamic MTs in et al., 1997; Ringhoff and Cassimeris, 2009). STX1C might be a the cells (Bunker et al., 2004). However, given that STX1C sequestering protein, in a manner similar to that of stathmin. decreased not only dynamic MT density but also total MT We also demonstrated that alterations in MT instability, through the effect of the STX1C TBD, affected the motility of vesicles containing GLUT1 protein as well as their translocation to the PM, consistent with a previous report (Marchant et al., 2002). Although this effect on VT motility might directly reflect the state of individual MTs, such as destabilization and disruption, it is also possible that alterations in the association between vesicles and MTs could affect VT motility. The STX

Journal of Cell Science family of molecules, which now consists of 19 members, regulates every process of membrane transport between intracellular compartments and delivery to the target organelles (Teng et al., 2001). GLUT1 vesicles are known to interact with cytoskeletal proteins, such as myosin VI, a-actinin-1 and the kinesin-1 family member 1B (KIF-1B) through GLUT1CBP (also known as TIP2 and GIPC1), resulting in targeting of GLUT1 to specific subcellular sites, either by tethering the transporter to cytoskeletal motor proteins or by anchoring it to the actin cytoskeleton (Bunn et al., 1999; Reed et al., 2005), whereas syntaxin has not been shown to participate in GLUT1-vesicle transport. Recently, it has been reported that the translocation of GLUT4, a member of the facilitative GLUT family which includes GLUT1, depends on syntaxins and the cytoskeletal system (Dransfeld et al., 2001; Perera et al., 2003; Pooley et al., 2008; Tong et al., 2001; Wang et al., 1998). For example, STX6, 8 and 12 colocalize on the GLUT4 vesicle, whereas STX6 regulates transport of the GLUT4 vesicle in a membrane-trafficking step that sequesters GLUT4 away from traffic destined for the PM (Perera et al., 2003). Fig. 7. Quantification of endocytosed and recycled GLUT1. Centromere protein F (CENPF), which provides a link between (A,B) Quantification of endocytosed and recycled GLUT1 using FCM in cells proteins of the SNARE system, including STX4, associates stably expressing exogenous constructs. Endocytosed (A) or recycled (B) GLUT1–antibody complexes were quantified using the Qdot705-tagged directly the MT network, resulting in the regulation of GLUT4 secondary antibody for FCM analysis. Negative controls (parent; Pt) without vesicular transport (Pooley et al., 2008). In neurons, syntabulin, a signals of Qdot705 and DsRed are shown in the upper right panel figure. A motor-protein-linked factor associated with both STX1A and histogram of Qdot705 intensity in double-positive cells gated with DsRed and KIF5B has also been implicated in the anterograde axonal Qdot705 signal based on the control threshold is shown in the lower panels. transport of STX1A-containing vesicles on the MT network (Cai 828 Journal of Cell Science 125 (4)

Table 6. Effect of STX1C-TBD on the GLUT1 intensity endocytosed and recycled to PM Endocytosed Recycled Recyc/Endo MFI % (vs Cont.) MFI % (vs Cont.) Ratio (vs Cont.) Pt 62.7623.3 100 24.265.1 100 1.00 Vec 47.9612.0 87.069.4 21.563.5 93.965.4 1.08 WT 34.367.3**,{{ 74.569.6**,{ 15.462.4*,{{,{{ 65.766.0*,{ 0.88 AAAA 44.9610.7 88.067.8 18.163.2 98.064.4 1.11

The GLUT1 intensity is given as mean values of fluorescent intensity (MFI) from FCM assays. A minimum of 10,000 cells was analyzed in each FCM assay. Value are the mean 6 s.e.m. Statistical significance was determined using a t-test with equal variance as follows: *P,0.01, **P,0.05 vs Vec; {P,0.01, {{P,0.05 vs AAAA; {{P,0.05 vs Pt. Cont., control.

et al., 2007; Su et al., 2004). Although FRSK cells do not express eluted with Laemmli buffer containing 10 mM EDTA. The blotted membrane were probed with a rabbit anti-STX1C polyclonal antibody (Nakayama et al., STX1A, it is possible that other PM syntaxins colocalize on 2003) or a mouse anti-b-tubulin monoclonal antibody (DM1A; Sigma). GLUT1 vesicles, as in the case of GLUT4. For example, a For GLUT immunoblotting, a total cell membrane preparation was made as ubiquitously distributed motor-protein-linked factor for STX2 and described previously (Nakayama et al., 2004). Immunoblotted signals were STX3, such as PM syntaxin, might be a candidate GLUT1 detected using the ECL system (Amersham) and were quantified using LAS-3000 (Fujifilm). colocalizing protein and a competitor of STX1C, because the amino acid sequence of the TBD and the SNARE motif was Measurement of glucose uptake particularly highly conserved between them (Teng et al., 2001). Glucose transport was assayed by measuring the uptake of [3H]2-deoxy-D-glucose Alternatively, it has been reported that STX2 and STX3 bind to (2-DG), essentially as described previously (Nakayama et al., 2004). The 2-DG Munc18b, which is located along MTs and affects granule uptake was linear between 0 and 30 minutes of incubation.

associated with the MT network in mast cells Imaging of FM1-43 and FM4-64 vesicles (Martin-Verdeaux et al., 2003). It is also possible that STX1C After FRSK parent cells were transfected with DsRed constructs, cells were plays a role as a binding competitor with Munc18b and STX2 or incubated with 5 nM FM1-43 or FM4-64 (both from Molecular Probes) for 1 hour STX3 in the MT network, because STX1C has the ability to bind to at 37˚C. All experiments were done at 37˚C. After incubation, cells were washed twice with DMEM and incubated with HEPES-buffered DMEM containing 10% Munc18b (Jagadish et al., 1997). Further studies of motor-protein- FCS for 30 minutes at 37˚C. After incubation, cells were observed using a time- linked factors and the function of Munc18b with STX1C within the lapse video microscopy system. MT network could help provide an understanding of the regulatory mechanism of STX1C in GLUT1-vesicle transport. Time-lapse video microscopy assay for examining dynamics of microtubules and vesicle transport Taken together, our results suggest a STX1C-TBD-dependent Analysis of microtubule (MT) dynamics and vesicle-transport (VT) motility was mechanism for the regulation of GLUT1 translocation and carried out according to previous studies with slight modifications (Bunker et al., GLUT1-vesicle transport, providing important insights into the 2006; Szodorai et al., 2009). MT dynamics and VT experiments were done using FRSK cells stably expressing a GFP–a-tubulin and the FRSK parent cell line, Journal of Cell Science physiological function and the mechanism of soluble syntaxins, respectively. Cells were grown on 35-mm culture dishes and observed in a control including STX1C. chamber equipped with a microscope maintained at 3760.5˚C by an MI-IBC heat regulator (Olympus). For MT and vesicle dynamics experiments, cell images were Materials and Methods obtained at 2.13- to 3.15-second intervals on an IX71 microscope (Olympus) and Plasmid constructs an ORCA-ER cooled CCD camera (Hamamatsu). EGFP and FM1-43 were excited For the expression of DsRed monomer fusion protein, the STX1C open reading through a 485DF15 filter (Omega Opticals, Brattleboro, VT) and their emissions frame (AB086954M) was subcloned in-frame into the pDsRed-C1 expression were collected through 540/50BP filters (Chroma). DsRed and FM4-64 were vector (Clontech) for time-lapse image analysis and into the pPro Ex-1 vector excited through a 575DF25 filter (Omega Opticals) and their emissions were (Invitrogen) for the in vitro binding assay. Mutated STX1C expression vectors collected through a 624/40BP filter (Semrock, Lake Forest, IL). MT ends and were constructed from the pDsRed–STX1C expression plasmid using the KOD- transport vesicles at the lamellar edge of interphase cells were imaged using the Plus Mutagenesis kit (Toyobo, Osaka, Japan). All the constructs were verified by AquaCosmos image analysis software (Hamamatsu). sequencing. To evaluate the motility of MT-dependent secretory vesicles, with the exception of short-range motions such as vesicle rotation and oscillation, only FM1-43 vesicles anterogradely moving towards the PM in the flat peripheral region were Cell culture and transfection analyzed until the vesicle was seen to move in another direction, switch to short- The rat epithelial cell line, FRSK, was purchased from the Health Science range motion, or disappear. At least 38 randomly selected MTs from more than 12 Research Resources Bank (HSRRB; Osaka, Japan). Cells were maintained independent cells or at least 37 randomly selected transport vesicles from more in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen), than four independent cells were observed. The distance traveled from a point of supplemented with 10% (v/v) FCS (Sigma), penicillin (100 mg/ml) and origin (from a pause point to the next pause point) and the velocity of individual streptomycin (100 mg/ml). For time-lapse analysis, cells were plated on poly-D- MT ends and transport vesicles were determined using the distance measurement lysine-coated 35-mm glass bottom micro-well dishes and cultured in HEPES- command on the AquaCosmos image analysis software. These values were buffered Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing transferred to a Microsoft Excel spreadsheet and used to analyze the dynamics of 10% FCS at 37˚Cin5%CO2. For transfection, cells were treated with a mixture individual MTs and vesicles. Changes in MT length greater than 0.5 mm were of plasmid DNA and LipofectAMINE-2000 reagent (Invitrogen). For stable designated as growth or shortening events. Periods in which length changes were transformants of GFP–a-tubulin or DsRed derivatives, cells were selected with less than 0.5 mm were designated as phases of attenuated MT dynamics (pause). G418 (Sigma) and cloned. The fraction of time spent was calculated as the time that MTs spent in three phases (growth, shortening, pausing) relative to the total time tracked. The density In vitro protein binding study and immunoblot analysis of dynamic MTs and total MTs was calculated as the number of MTs divided by In vitro binding studies were carried out according to our previous method with the total area measured. We operationally defined dynamic MTs as those grown or slight modifications (Fujiwara et al., 1997; Itoh et al., 1999). Briefly, the purified shortened without a pause phase of more than a quarter of the lifespan (measured tubulin fraction was incubated with 6His-STX1C-linked Ni+ resin in the presence in minutes) of an individual MT. The percentage of dynamic MTs was calculated of various concentrations of synthetic peptides (Biosynthesis). After washing with as the number of dynamic MTs divided by the total number of MTs. Dynamicity the binding buffer [10 mM HEPES (pH 7.6), 150 mM KCl, 0.1% protease was calculated as the time spent growing and shortening divided by the total time inhibitors cocktail, 1% phosphatase inhibitor cocktail], the bound material was measured. STX1C regulates the GLUT1 recycling phase 829

Dual time-lapse video imaging of vesicle transport on microtubules Statistical analyses FRSK cells stably expressing GFP–a-tubulin were loaded with 5 nM FM4-64 for The data are expressed as means 6 s.e.m. and were analyzed statistically using 1 hour at 37˚C and then washed extensively in DMEM. GFP–a-tubulin and FM4-64 Microsoft Excel and Prism software (GraphPad Software). P-values of less than were simultaneously excited (492DF18 filter; Omega Opticals). Their emissions 0.05 were considered to be significant. were detected with a DualView setup (Optical Insights) with appropriate filters (dichroic filter 565cdxr, emission filter 670DF40) and projected onto the two halves Acknowledgements of the CCD-EM chip (Hamamatsu). Image sequences were acquired in the time- lapse mode at 2.5- to 3-second intervals on an IX81 microscope (Olympus) using a We express our gratitude to M. Sanada (Kyorin University) for 1006 Olympus objective lens (numerical aperture, 1.4) and analyzed using the experimental assistance. We are greatly indebted to T. Tojima, H. MetaMorph imaging software (Molecular Devices). Akiyama, R. Itofusa and K. Ohtawa (RIKEN-BSI) for advice on the measurement of MT dynamics and VT motility, measurement with Immunocytochemistry the DualView system, histochemical experiments and FCM analysis, Translocation of GLUT1 in FRSK cells was observed as described previously respectively. (Nakayama et al., 2004). Double staining of FM1-43FX and b-tubulin was carried out according to a previous report (Tojima et al., 2007). Images were obtained Funding using a confocal scanning laser microscope, FV-1000 (Olympus) that was equipped with a triple band-pass filter set. This study was supported by grants-in-aid from the Japan Society for Internalized and recycled GLUT1 in FRSK cells was visualized as described the Promotion of Science for Japanese Junior Scientists [grant previously (Kamiguchi et al., 1998). For the internalization assay, cells were number 170679 to T.N.]; and the Ministry of Education, Culture, incubated with mouse monoclonal anti-GLUT1 (40 mg/ml) (R&D Systems) for 30 Sports, Science and Technology in Japan [Scientific Research (B) minutes at 37˚C to allow for endocytosis of the IgG bound to GLUT1 on the PM. (grant number 19300133) to K.A.]. After rinsing at 4˚C, the cells were fixed with 4% formaldehyde for 30 minutes. Because this fixation protocol did not permeabilize the cells, subsequent incubation with unlabeled anti-mouse IgG (400 mg/ml; Jackson Laboratory) for Supplementary material available online at 30 minutes at 37˚C specifically blocked the cell-surface IgG. Then, the cells were http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.081943/-/DC1 fixed again with 4% formaldehyde for 10 minutes to immobilize the unlabeled secondary antibody. After washing, the cells were permeabilized and blocked with 0.1% Triton X-100 and 10% horse serum in PBS for 30 minutes. Internalized References GLUT1 was visualized by incubating the cells with Alexa-Fluor-488-conjugated Bennett, M. K., Calakos, N. and Scheller, R. H. (1992). Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, anti-mouse IgG (1:100) for 30 minutes at 20˚C. For analysis of endocytosed 255-259. GLUT1 vesicles, the density of vesicles was calculated by counting positive Brumback, A. C., Lieber, J. L., Angleson, J. K. and Betz, W. J. (2004). Using FM1- signals existing in at least 10 rectangle regions in a transfected cell. The average 43 to study neuropeptide granule dynamics and exocytosis. Methods 33, 287-294. density was calculated in at least 81 cells. Bunker, J. M., Wilson, L., Jordan, M. A. and Feinstein, S. C. (2004). Modulation of For double staining with endocytic vesicles, cells were cultured on a gridded microtubule dynamics by tau in living cells: implications for development and slide dish. Mouse monoclonal anti-GLUT1 (40 mg/ml) along with 10 mM FM1- neurodegeneration. Mol. Biol. Cell 15, 2720-2728. 43FX (Invitrogen) were taken up by living cells for 30 minutes at 37˚C, followed Bunker, J. M., Kamath, K., Wilson, L., Jordan, M. A. and Feinstein, S. C. (2006). by destaining with DMEM containing 1 mM ADVASEP-7 (Biotium, Hayward, FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics CA) for 10 minutes at 37˚C. After cells were fixed with 4% formaldehyde, FM1- in cells. J. Biol. Chem. 281, 11856-11863. 43FX images were obtained. GLUT1 IgGs on the surface of the cells were blocked Bunn, R. C., Jensen, M. A. and Reed, B. C. (1999). Protein interactions with the with unlabeled anti-mouse IgG (400 mg/ml) and permeabilized with a 0.1% Triton glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 X-100. After blocking with 10% goat serum, cells were visualized with an Alexa- and the cytoskeleton. Mol. Biol. Cell 10, 819-832. Fluor-594-conjugated anti-mouse IgG. GLUT1 signals were then obtained. Cai, Q., Pan, P. Y. and Sheng, Z. H. (2007). Syntabulin-kinesin-1 family member 5B- For the recycling assay, cells were incubated with mouse monoclonal anti- mediated axonal transport contributes to activity-dependent presynaptic assembly. J. Neurosci. 27, 7284-7296.

Journal of Cell Science GLUT1 (40 mg/ml) for 30 minutes at 37 C to allow for endocytosis of the GLUT1– ˚ Desai, A. and Mitchison, T. J. (1997). Microtubule polymerization dynamics. Annu. IgG complex. The cells were cooled to 4˚C and incubated with unlabeled anti- Rev. Cell Dev. Biol. 13, 83-117. mouse IgG (400 mg/ml) for 30 minutes at 4˚C to block the cell-surface IgG. After Dransfeld, O., Rakatzi, I., Sasson, S., Gruzman, A., Schmitt, M., Haussinger, D. and extensive washes at 4˚C, the cells were incubated at 37˚C for various periods in Eckel, J. (2001). Eicosanoids participate in the regulation of cardiac glucose transport prewarmed 10% FCS–DMEM. This incubation allowed the cells to recover and by contribution to a rearrangement of actin cytoskeletal elements. Biochem. J. 359, proceed with the trafficking of endocytosed GLUT1 that had been tagged with the 47-54. anti-GLUT1 IgG. The cells were then fixed with 4% formaldehyde, and recycled Fujiwara, T., Yamamori, T., Yamaguchi, K. and Akagawa, K. (1997). Interaction of GLUT1 on the cell surface was detected by visualizing the anti-GLUT1 IgG that HPC-1/syntaxin 1A with the cytoskeletal protein, tubulin. Biochem. Biophys. Res. had not been blocked with the unconjugated secondary antibody. This was done by Commun. 231, 352-355. incubating the unpermeabilized cells with Alexa-Fluor-488-conjugated anti-mouse Howard, J. and Hyman, A. A. (2009). Growth, fluctuation and switching at IgG (1:100) for 30 minutes at 20˚C. Images were obtained using an ORCA-ER microtubule plus ends. Nat. Rev. Mol. Cell Biol. 10, 569-574. camera. In internalization and recycling experiments, Alexa-Fluor-488-conjugated Ibaraki, K., Horikawa, H. P., Morita, T., Mori, H., Sakimura, K., Mishina, M., secondary antibody did not recognize newly synthesized GLUT1 transported into Saisu, H. and Abe, T. (1995). Identification of four different forms of syntaxin 3. the PM from the soma because it was not bound to the IgG. We operationally Biochem. Biophys. Res. Commun. 211, 997-1005. defined the cell recycled GLUT1 on the PM as that showing continuous GLUT1 Inoue, A., Obata, K. and Akagawa, K. (1992). Cloning and sequence analysis of signals on more than 12.5% of the cell circumference. cDNA for a neuronal cell membrane antigen, HPC-1. J. Biol. Chem. 267, 10613- 10619. Itoh, T., Fujiwara, T., Shibuya, T., Akagawa, K. and Hotani, H. (1999). Inhibition of Flow cytometry (FCM) analysis of internalized and recycled GLUT1 microtubule assembly by HPC-1/syntaxin 1A, an exocytosis relating protein. Cell FCM analysis of internalized and recycled GLUT1 was carried out according to a Struct. Funct. 24, 359-364. previous study with slight modifications (Reddy et al., 2001). FRSK cells stably Jagadish, M. N., Tellam, J. T., Macaulay, S. L., Gough, K. H., James, D. E. and expressing plasmids encoding DsRed derivatives were used for FCM analysis. After Ward, C. W. (1997). Novel isoform of syntaxin 1 is expressed in mammalian cells. the cells were trypsinized, floating cells were neutralized with excess FCS and Biochem. J. 321, 151-156. incubated with mouse monoclonal anti-GLUT1 (40 mg/ml) for 30 minutes at 37˚C Jahn, R. and Sudhof, T. C. (1999). Membrane fusion and exocytosis. Annu. Rev. to allow for endocytosis of the GLUT1–IgG complex. According to the Biochem. 68, 863-911. immunocytochemical procedures for internalized and recycled GLUT1, the Jerdeva, G. V., Wu, K., Yarber, F. A., Rhodes, C. J., Kalman, D., Schechter, J. E. GLUT1 signal was detected with Qdot705-conjugated anti-mouse IgG antibody. and Hamm-Alvarez, S. F. (2005). Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells. J. Cell Sci. 118, Soaking, washing and fixation were conducted with TBS buffer. For confirmation of 4797-4812. the GLUT1 signal, a portion of the prepared cells was visualized with Alexa-Fluor- Jourdain, L., Curmi, P., Sobel, A., Pantaloni, D. and Carlier, M. F. (1997). Stathmin: 488-conjugated anti-mouse IgG antibody and examined microscopically. Cells a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin labeled with Qdot705 were analyzed using a FACSAria II (BD Bioscience). DsRed molecules. Biochemistry 36, 10817-10821. was excited with a 532-nm laser line (diode-pumped solid state laser) and its Kamiguchi, H., Long, K. E., Pendergast, M., Schaefer, A. W., Rapoport, I., emission was collected through a 610/20 BP filter. Qdot705 was excited with a UV Kirchhausen, T. and Lemmon, V. (1998). The neural cell adhesion molecule L1 laser at 355 nm, and the fluorescence signal was collected through 670LP filter. The interacts with the AP-2 adaptor and is endocytosed via the -mediated pathway. data were analyzed using FlowJo software (Tree Star; www.freestar.com). J. Neurosci. 18, 5311-5321. 830 Journal of Cell Science 125 (4)

Kinet, S., Swainson, L., Lavanya, M., Mongellaz, C., Montel-Hagen, A., Craveiro, M., Pooley, R. D., Moynihan, K. L., Soukoulis, V., Reddy, S., Francis, R., Lo, C., Ma, Manel, N., Battini, J. L., Sitbon, M. and Taylor, N. (2007). Isolated receptor binding L. J. and Bader, D. M. (2008). Murine CENPF interacts with syntaxin 4 in the domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ regulation of vesicular transport. J. Cell Sci. 121, 3413-3421. T cells. Retrovirology 4,31. Quinones, B., Riento, K., Olkkonen, V. M., Hardy, S. and Bennett, M. K. (1999). Lewis, S. A., Wang, D. H. and Cowan, N. J. (1988). Microtubule-associated protein Syntaxin 2 splice variants exhibit differential expression patterns, biochemical MAP2 shares a microtubule binding motif with tau protein. Science 242, 936-939. properties and subcellular localizations. J. Cell Sci. 112, 4291-4304. Manneville, J. B., Etienne-Manneville, S., Skehel, P., Carter, T., Ogden, D. and Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasma membrane repair is Ferenczi, M. (2003). Interaction of the actin cytoskeleton with microtubules regulates mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell 106, 157-169. secretory organelle movement near the plasma membrane in human endothelial cells. Reed, B. C., Cefalu, C., Bellaire, B. H., Cardelli, J. A., Louis, T., Salamon, J., J. Cell Sci. 116, 3927-3938. Bloecher, M. A. and Bunn, R. C. (2005). GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evidence supporting an adapter function for Marchant, J. S., Subramanian, V. S., Parker, I. and Said, H. M. (2002). Intracellular GLUT1CBP. Mol. Biol. Cell 16, 4183-4201. trafficking and membrane targeting mechanisms of the human reduced folate carrier Ringhoff, D. N. and Cassimeris, L. (2009). Stathmin regulates centrosomal nucleation in Mammalian epithelial cells. J. Biol. Chem. 277, 33325-33333. of microtubules and tubulin dimer/polymer partitioning. Mol. Biol. Cell 20, 3451- Martin-Verdeaux, S., Pombo, I., Iannascoli, B., Roa, M., Varin-Blank, N., Rivera, J. 3458. and Blank, U. (2003). Evidence of a role for Munc18-2 and microtubules in mast cell Rothman, J. E. (1994). Mechanisms of intracellular protein transport. Nature 372,55- granule exocytosis. J. Cell Sci. 116, 325-334. 63. Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Sahlen, G. E., Egevad, L., Ahlander, A., Norlen, B. J., Ronquist, G. and Nilsson, Allard, W. J., Lienhard, G. E. and Lodish, H. F. (1985). Sequence and structure of B. O. (2002). Ultrastructure of the secretion of prostasomes from benign and a human glucose transporter. Science 229, 941-945. malignant epithelial cells in the prostate. Prostate 53, 192-199. Nakayama, T., Fujiwara, T., Miyazawa, A., Asakawa, S., Shimizu, N., Shimizu, Y., Simonsen, A., Bremnes, B., Ronning, E., Aasland, R. and Stenmark, H. (1998). Mikoshiba, K. and Akagawa, K. (1997). Mapping of the human HPC-1/syntaxin 1A Syntaxin-16, a putative Golgi t-SNARE. Eur. J. Cell Biol. 75, 223-231. gene (STX1A) to 7 band q11.2. Genomics 42, 173-176. Singh, S. P., Gao, Y., Singh, L. D., Kunapuli, S. P. and Ravindra, R. (1998). Role of Nakayama, T., Matsuoka, R., Kimura, M., Hirota, H., Mikoshiba, K., Shimizu, Y., microtubules in glucose uptake by C6 glioma cells. Pharmacol. Toxicol. 83, 83-89. Shimizu, N. and Akagawa, K. (1998). Hemizygous deletion of the HPC-1/syntaxin Su, Q., Cai, Q., Gerwin, C., Smith, C. L. and Sheng, Z. H. (2004). Syntabulin is a 1A gene (STX1A) in patients with Williams syndrome. Cytogenet. Cell Genet. 82, 49- microtubule-associated protein implicated in syntaxin transport in neurons. Nat. Cell 51. Biol. 6, 941-953. Nakayama, T., Mikoshiba, K., Yamamori, T. and Akagawa, K. (2003). Expression of Szodorai, A., Kuan, Y. H., Hunzelmann, S., Engel, U., Sakane, A., Sasaki, T., Takai, Y., syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, is enhanced by Kirsch, J., Muller, U., Beyreuther, K. et al. (2009). APP anterograde transport requires phorbol-ester stimulation in astroglioma: participation of the PKC signaling pathway. Rab3A GTPase activity for assembly of the transport vesicle. J. Neurosci. 29, 14534- FEBS Lett. 536, 209-214. 14544. Nakayama, T., Mikoshiba, K., Yamamori, T. and Akagawa, K. (2004). Activation of Teng, F. Y., Wang, Y. and Tang, B. L. (2001). The syntaxins. Genome Biol. 2, REVIEWS3012. syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, by phorbol 12- Tojima, T., Akiyama, H., Itofusa, R., Li, Y., Katayama, H., Miyawaki, A. and myristate 13-acetate (PMA) suppresses glucose transport into astroglioma cells via Kamiguchi, H. (2007). Attractive axon guidance involves asymmetric membrane the glucose transporter-1 (GLUT-1). J. Biol. Chem. 279, 23728-23739. transport and exocytosis in the growth cone. Nat. Neurosci. 10, 58-66. Olson, A. L. and Pessin, J. E. (1996). Structure, function, and regulation of the Tong, P., Khayat, Z. A., Huang, C., Patel, N., Ueyama, A. and Klip, A. (2001). mammalian facilitative glucose transporter gene family. Annu. Rev. Nutr. 16, 235- Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell 256. membrane ruffles. J. Clin. Invest. 108, 371-381. Pereira, S., Massacrier, A., Roll, P., Verine, A., Etienne-Grimaldi, M. C., Poitelon, Y., Wang, Q., Bilan, P. J., Tsakiridis, T., Hinek, A. and Klip, A. (1998). Actin filaments Robaglia-Schlupp, A., Jamali, S., Roeckel-Trevisiol, N., Royer, B. et al. (2008). participate in the relocalization of phosphatidylinositol3-kinase to glucose transpor- Nuclear localization of a novel human syntaxin 1B isoform. Gene 423, 160-171. ter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 Perera, H. K., Clarke, M., Morris, N. J., Hong, W., Chamberlain, L. H. and Gould, adipocytes. Biochem. J. 331, 917-928. G. W. (2003). Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes. Mol. Biol. Wittmann, T. and Waterman-Storer, C. M. (2001). Cell motility: can Rho GTPases Cell 14, 2946-2958. and microtubules point the way? J. Cell Sci. 114, 3795-3803. Journal of Cell Science