Methanol fixation allows better visualization of Kal7. To compare methods for

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1 Supplementary Data Methanol fixation allows better visualization of Kal7. To compare methods for visualizing Kal7 in dendrites, mature cultures of dissociated hippocampal neurons (DIV21) were fixed with 4% paraformaldehyde or cold methanol, and triple-stained with antibodies to Kal7, VGLUT1 and MAP2 (Fig. S1). Methanol fixation yielded more intense Kal7 and VGLUT1 staining in puncta arrayed along dendrites (Fig. S1A). With the same exposure time, paraformaldehyde fixation yielded almost no VGLUT1 staining and Kal7 staining was diminished (Fig. S1C). Longer exposure times revealed some staining after paraformaldehyde fixation (Fig. S1D). Pre-absorption of the Kal7 antibody with its antigenic peptide eliminated Kal7 staining (Fig. S1C). Kal7GFP is bioactive. Expression of Kal7 in interneurons, which are not rich in spines, suggested a different role for Kal7. To explore this possibility, we developed a bioactive GFP-tagged version of Kal7. In addition to allowing live cell imaging, the GFP tag allows use of different antisera. Since appending GFP to the COOH-terminus of Kal7 would disrupt its PDZ binding motif, GFP was appended to the NH 2 -terminus, in place of the myc tag, or inserted into predicted loop regions within spectrin repeats 5 or 9 (Fig. S2A). Each of the GFP-tagged variants of Kal7 produced massive lamellipodia and activated Rac when expressed in fibroblasts, but appending GFP to the NH 2 -terminus of Kal7 or inserting GFP into spectrin repeat 5 yielded aggregates of fluorescent protein (data not shown). Kal7 with EGFP(A206K) inserted into spectrin repeat 9 did not form large aggregates when expressed in hippocampal neurons and was used in all subsequent studies (referred to as Kal7GFP) (Fig. S2A).

2 To compare the properties of Kal7 and Kal7GFP, peak RAPID cells were transfected with vectors encoding both proteins; cell lysates were separated into soluble, TX-100-solubilized and insoluble fractions. Western blot analysis demonstrated that a small fraction of the Kal7 and Kal7GFP was soluble, with the majority of both proteins recovered in the TX-100 insoluble cytoskeletal fraction (Fig. S2M). To determine whether Kal7GFP is an active GEF, peak RAPID cells were transfected with vectors encoding KalGEF1, Kal7 or Kal7GFP. After 48 h, cells were extracted for assessment of Rac activation using the Pak-CRIB domain-binding assay (Schiller et al., 2005). Expression of each protein resulted in activation of Rac (Fig. S2C). Expression of Kal7 induces spine formation in cortical neurons (Penzes et al., 2001b). To compare the ability of Kal7 and Kal7GFP to induce spine formation, freshly dissociated hippocampal neurons were transfected with vectors encoding GFP alone, Kal7GFP and MycKal7; at DIV16, cells were doubly stained with antibodies to GFP (Fig. S2D, G, J) or myc (Fig. S2B) and Kal7 (Fig. S2E, H, K, N). In aspiny neurons expressing GFP, staining filled the dendritic shaft (Fig. S2D); GFP appeared in the spines, filopodia, and dendritic shafts of spiny neurons (Fig. S2G). Since the staining patterns for MycKal7 and Kal7GFP are identical, we conclude that Kal7GFP is a valid marker to identify dendritic spines. Expression of Kal7GFP or Kal7 in hippocampal pyramidal neurons results in the formation of an increased number of dendritic spines (Fig. S2J, M). This established that Kal7GFP is biologically active in neurons, as in fibroblasts. Based on these observations, we decided to use Kal7GFP to probe Kal7 function in interneurons. Since we did not want levels of exogenous Kal7GFP to greatly exceed levels of endogenous Kal7, we

3 quantified the increase in Kal7 staining observed following expression of Kal7GFP. To determine Kal7 in interneurons, cells were triple-stained with antibodies to GFP (Fig. S2P, T), GAD65 (Fig. S2Q, U) and Kal7 (Fig. S2R, V). Kal7 staining in neurons expressing Kal7GFP was about 2-fold higher than Kal7 staining in non-transfected neighboring neurons (Fig. S2P-W). Kal7 and Kal7GFP have similar effects on spine formation and GFP is an adequate marker of dendritic spines. Dissociated DIV1 hippocampal neurons were transfected with vector encoding soluble GFP alone, co-transfected with vectors encoding soluble GFP and MycKal7, or transfected with vector encoding Kal7GFP alone. Cells were triple-stained at DIV15 with antibodies to GFP, GAD65/67 or GAD65 and myc or MAP2. No spines were found on the dendrites of GAD65/67 positive interneurons expressing GFP alone (Fig. S3A, D). GFP-filled spines were observed in GAD65/67 positive interneurons expressing GFP and MycKal7 and visualized using antibody to GFP or to Myc (Fig. S3B, E). Myc staining is localized to the tips of GFP positive spines (arrows in Fig. S3E); large spine heads and a long fine neck are apparent. Importantly, linear spine densities determined using GFP or Myc staining are indistinguishable (98% overlap, 300 spines counted in each neuron, n=10). In addition, linear spine density is identical in hippocampal interneurons expressing Kal7GFP or MycKal7 (Fig. S3G); as observed with Myc staining of MycKal7, expression of Kal7GFP yields GFP positive clusters at the tips of spines (Fig. S3F) Expression of Kal7GFP causes spine formation in hippocampal interneurons. The dendrites of interneurons expressing GFP are almost free of spine-like structures, and spines are never found on the soma of interneurons expressing GFP (Fig. S4A).

4 Expression of Kal7GFP induces a marked increase in spine-like structures along the dendrites and on the soma of interneurons (Fig. S4E, H, I). In GFP-expressing interneurons, GFP is diffusely merged with staining for MAP2 (Fig. S4D). In contrast, in Kal7GFP-expressing interneurons, the GFP occurs in clusters both within the dendritic shaft and at the tips of dendritic spines (Fig. S4H, I). R7-Kal7CT enters neurons and binds to dendrites. Neurons incubated with the R7- Kal7CT peptide (10µM) and stained with polyclonal Kal7 antibody exhibited readily detectable staining in the soma, dendrites and synaptic structures, indicating intracellular peptide uptake (Fig. S5A-E, H). Neurons incubated with R7-Kal7CTmutant peptide showed staining in the cell soma (Fig. S5F), with low levels in dendrites. Endogenous Kal7 was visualized in the soma in cultures treated with vehicle (Fig. S5G). Polyclonal Kal7 antibody staining in dendrites is dependent on the concentration of Kal7CT peptide (data not shown). Staining of R7-Kal7CT was maximal 1 h after washing out the peptide; levels then decreased gradually, remaining detectable for 3 days in a small population of neurons (not shown). Supplementary Figures Fig. S1. Effect of fixation on visualization of synaptic Kal7. Cultured hippocampal neurons (DIV 21) were fixed with methanol (-20 C) for 12 min (A-B) or 4% paraformaldehyde (C-D) for 18 min. Neurons were triple-stained with antibodies specific Kal7 (red), VGLUT1 (green) and MAP2 (blue). The same exposure time was used for A and C. D, longer exposure time. Blocking the Kal7 antibody with its antigenic peptide abolished staining (B).

5 Fig. S2. Kal7GFP is bioactive. (A) GFP was inserted into a putative loop region in spectrin repeat 9 of Kal7. (B) peak RAPID cells were transfected with vectors encoding Kal7 or Kal7GFP; Western blot analysis (myc, left; GFP, right) demonstrates that both proteins fractionate in a similar manner (Sol, soluble; TX, TX-100 solubilized; P, SDS solubilized pellet). (C) peak RAPID cells expressing KalGEF1, Kal7 or Kal7GFP were extracted for measurement of Rac-GTP using GST-Pak-CRIB domain resin. Inputs, myc antibody (top), Rac bound to resin, Rac antibody (bottom). Dissociated hippocampal neurons (DIV1) were transfected with vectors encoding GFP (D, G, P), Kal7GFP (J, T) or myc-tagged Kal7 (M); neurons were fixed at DIV18 and visualized with monoclonal antibodies specific for GFP (D, G, J) or myc (N) and polyclonal antibody specific for Kal7 (E, H, K, N). In aspiny dendrites, endogenous Kal7 is localized to dendritic shafts (E-F). In spiny dendrites, endogenous Kal7 is localized to spine tips (arrow heads) or dendritic shafts (arrows) (H-I). Kal7 staining overlapped GFP (L) or myc (O) staining in Kal7GFP and Kal7 transfected dendrites, respectively. Expression of Kal7 and Kal7GFP caused the formation of similar spine-like structures. P-W, Neurons fixed with 4% paraformaldehyde were triple-stained with antibodies to GFP (green, P, T), GAD65 (blue, Q, U) and Kal7 (red, R, V). Kal7GFP positive neurons (n=15) showed a 1.9-fold increase in Kal7 staining compared with control neurons expressing GFP only (n=16). Fig. S3. Expression of Kal7 or Kal7GFP causes formation of similar spine-like structures. To determine whether Kal7 andkal7gfp have similar effects on spine formation and whether free GFP can be used to identify spines, dissociated DIV1 hippocampal neurons were transfected with vector encoding soluble GFP alone (A, D), co-transfected with vectors encoding soluble GFP and MycKal7 (B, E), or transfected

6 with vector encoding Kal7GFP alone (C, F). Cells were triple-stained at DIV15 with antibodies to GFP (A-C, green), GAD65/67 (A, B, blue) or GAD65 (C, blue) and myc (A, B, red) or MAP2 (C, red). GAD65 antibody produced much brighter staining at nerve terminals than GAD67 antibody. D-F, high power images from boxed dendrites in A-C, respectively. Spine densities were quantified based on staining for GFP (A-C) or Myc (B) (G, n=11-12). For cultures co-transfected with GFP plus MycKal7, spine density was quantified three ways: using GFP (viewed as green with the red channel turned off); using Myc (viewed as red with the green channel turned off); using both GFP and Myc (yellow, with both red and green channels turned on). The spines in neurons expressing Kal7GFP fusion were counted using only the green channel (G). Scale bar, 20µm in A- C; 5µm in D-F. Fig. S4. Expression of Kal7GFP causes spine formation in hippocampal interneurons. Hippocampal neurons in dissociated culture were transfected with vector encoding GFP alone (A-D) or Kal7GFP (E-H) at DIV1, and triple-stained at DIV16 with antibodies to GFP (A, E), MAP2 (B, F), and GAD65/67 (C, E). A MAP2 positive neuron not stained for GAD65/67 (arrows, B-C), is not an interneuron. I, high power image from soma of merged image. The boxed dendrites in D and H are shown in Fig. 6L, M. Fig. S5. R7-Kal7CT peptide is retained in cells. Dissociated hippocampal neurons (DIV 18) were incubated with R7-Kal7CT peptide (10 µm, A, E, H), R7-Kal7CTmutant peptide (10 µm, F, I), or vehicle (G) for 45 min. Sixty min after peptide was removed, neurons were fixed with 4% paraformaldehyde and double-stained with polyclonal antibody to Kal7 (dilution 1:2000) (A) and monoclonal antibody to Kal7 (B), or stained with polyclonal antibody to Kal7 only (E-I). R7-Kal7CT is recognized by the polyclonal,

7 but not by the monoclonal Kal7 antibody; R7-Kal7CTmutant is recognized by the polyclonal antibody. Images E-G, and H-I were collected under identical conditions.