sensitive VBSs Vh subdomains EF EF

Transcription

1 Tlin- Tlin-EGFP-His ABD Mehno- ABD2 ABD3 sensitive VBSs DD 6xHis EGFP Vh sudomins EGFP-Vinulin EGFP Vt EGFP-Vh EGFP tinin- CH CH2 SP SP SP SP EF EF mcherry--tinin- mcherry CH CH2 SP SP SP SP EF EF Supplementry Figure. Proteins used in the study. () Construts used in this study. In tlin, the domins nmed,, 2 nd 3 re the F, F, F2 nd F3 sudomins of the FERM (four-point-one, ezrin, rdixin, moesin) domin. ABD, tin inding domin. DD, dimeriztion domin. Vh, vinulin hed. Vt, vinulin til. CH, lponin homology domin. SP, spetrin repet. EF, EF hnd motif. () SDS-PAGE of the purified proteins used in this work stined with Coomssie lue.

2 Tlin-EGFP in diss (.u.) High ffinity Clpin-2 site Low ffinity Clpin-2 site 2 3 EGFP ABD Mehno- ABD2 ABD3 sensitive VBSs DD 6xHis Tlin-EGFP-6xHis Rod-DD-EGFP-6xHis Rod Hed DD-EGFP-6xHis WB nti-his Tlin-EGFP-6xHis Coomssie Blue Alex594-Atin d Control + Clpin-2 (/2) 6 Control 2 8 Clpin-2 (/2) 4 e Alex594-Atin f Tlin- High ffinity Clpin-2 site Tlin ABD2 N ABD3 C Supplementry Figure 2. Orienttion of tlin on the surfe. (). Domin orgniztion of humn tlin fused with EGFP nd tgged with 6His t its C- terminus (tlin-egfp-his). The sheme shows the two lpin-2 levge sites. The high ffinity site is loted etween Q3 nd Q4 in the linker region tht onnets the hed nd the rod. The low ffinity site is loted etween K2493 nd M2494 in the region tht links the C-terminl tin inding domin (ABD) nd the dimeriztion domin (DD). () Tlin-EGFP- His is inuted with vrying dilutions of lpin-2. Clevge produts were resolved y SDS-PAGE nd nlyzed y Western lotting using n nti-his ntiody (left pnel) nd stined with Coomssie lue (entrl pnel). The levge produts re indited on the right. () In the ssy desried in this study, the ddition of lpin-2 (/2) results in the loss of EGFP fluoresene in tlin-egfp-his-oted diss (levge of tlin-egfp-his) nd prevents the nhoring of ontrtile tomyosin strutures. Conditions:.5 µm tlin-egfp- His (in the oting retion),.4 µm Myosin II, 2.4 µm of G-tin (2% Alex594-leled). Sle r, 2 µm. (d) Quntifition of the fluoresene of tlin-egfp-his in the diss in the sene nd presene of lpin-2. Dt re men ± SD. Asene of lpin-2 n = 85, presene of lpin-2 n = 8. The P vlue ws lulted (P <.) using t-test. Dt 2

3 olleted in two independent experiments. (e) We ompred the ility of full-length tlin nd tlin (deleted from the N-terminl hed) to support the self-ssemly of tomyosin les in the reonstituted ssy. SDS-PAGE shows tht the proteins were present t the sme onentrtion in the oting retions (left pnel). No ontrtile tin strutures were oserved when the miroptterned surfe ws first inuted with tlin insted of full-length tlin (right pnels). Conditions:.5 µm full-length tlin or tlin (during the oting retion),.4 µm Myosin II, 2.4 µm of G-tin (2% Alex594-leled). Sle r, 2 µm. (f) Sheme showing tht the N-terminl prt of tlin is ound to the surfe while its C-terminl ABD nd/or its entrl ABD intert with tin filments. 3

4 Normlized fluoresene (.u.) Normlized fluoresene (.u.) preleh.9.8 postleh s s 5 s d Time (s) preleh postleh s 2 s 5 s Time (s) Supplementry Figure 3. Fluoresene reovery fter photolehing (FRAP) of EGFP- Vh ound to tlin-oted diss FRAP experiments were performed to mesure the rte onstnt of fluoresene reovery of EGFP-Vh ound to tlin t stedy stte when onstnt fore is pplied in the presene of myosin nd short tin filments pped t their red ends. Conditions: µm of EGFP-Vh,.2 µm Myosin II, 2.4 µm of short tin filments pped t their red end. We used spinning disk onfol mirosope (-) nd TIRF mirosope (-d) to photoleh the smple nd monitor the fluoresene reovery. () In this representtive exmple, irulr region ( µm in dimeter) ws drwn round the left dis nd lehed y 2 itertive pulses of 2 ms eh using 49 nm lser t 5% power. Fluoresene reovery fter photolehing ws monitored for 5 s ( frme/ s). The fluoresene of the ontrol nonlehed dis (right) did not hnge during the reovery of the lehed diss. The timelpse shows the preleh fluoresene, nd the postleh fluoresene reovery t time, nd 5 s. Sle r, µm. () Kinetis showing the fluorersene reovery. Dt re men ± SD, n = 6. Dt olleted in two independent experiments. () In the representtive exmple, the surfe ws lehed y ms ontinuous illumintion using the evnesent wve generted y the 473 nm lser of TIRF mirosope. Fluoresene reovery fter photolehing ws monitored for 5 s ( frme/ s). The timelpse shows the preleh fluoresene, nd the postleh fluoresene reovery t time, 2 nd 5 s. Sle r, µm. Supplementry Movie 5. (d) Kinetis showing the fluoresene reovery. Dt re men ± SD, n = 28. Dt olleted in three independent experiments. 4

5 Frtion of EGFP-vinulin ound Light sttering intensity (.u.) Fluoresene (.u.) 3 EGFP-Vh (-T) tinin (-T) + tin (5 µm) -tinin (+T) + tin (5 µm) mcherry--tinin (-T) + tin (5 µm) mcherry--tinin (+T) + tin (5 µm) -tinin (-T) -tinin (+T) mcherry--tinin (-T) mcherry--tinin (+T) EGFP-Vh (+T) n.s tinin (mm) F-tin (µm) EGFP-Vinulin (µm) VBS (µm) EGFP- Vinulin F-tin K d =.5 µm VBS (µm) Supplementry Figure 4. Control of the tivity of -tinin, mcherry- -tinin, EGFP- Vh nd EGFP-vinulin () The rosslinking tivity of -tinin nd mcherry- -tinin ws ssessed y right ngle light sttering t 35 nm. -tinin nd mcherry- -tinin were inuted with 5 µm of MgATP-G-tin in polymerizing uffer (5mM Tris ph7.8, 25 mm KCl, mm MgCl 2,, mm CCl 2,.2 mm ATP, mm DTT). We mesured the inrese in light sttering etween t nd the finl stedy stte (h t room temperture). Both -tinin nd mcherry- -tinin indue dose dependent inrese in the light sttering intensity in the presene of tin, inditing the formtion of tin filment undles (irles). In the sene of tin, light sttering did not hnge signifintly during h (squres). Beuse the purifition of - tinin nd mcherry- -tinin used in this study inludes teril lysis step in the presene of Triton X, we verified tht the tivity of the proteins purified in the sene of Triton X (-T, open symols) nd in the presene of Triton X (+T, losed symols) re the sme. Two independent mesurements re shown for eh ondition. () The purifition of EGFP-Vh used in this study inludes teril lysis step in the presene of Triton X. EGFP-Vh purified in the sene (red r) or presene of Triton X (lue r) re reruited t the sme level in tlin oted diss t stedy stte when onstnt fore ws pplied in the presene of myosin nd short tin filments pped t their red ends. Dt re men ± SD. Asene of Triton n = 33, Presene of Triton n = 4. The P vlue ws lulted (P =.24) using t-test. Dt olleted in two independent experiments. The onditions re desried in the Fig. 2. () EGFP-vinulin inds to tin filments in tlin-dependent mnner. (Left) EGFP-vinulin (2 µm) ws inuted with F-tin (5 µm) in the presene of inresing onentrtions of tlin (VBS) s indited. After ultrentrifugtion, the pellet frtion ws nlyzed y SDS-PAGE nd stined with Coomssie lue. (Right) The frtion of EGFP-vinulin ound to tin ws plotted versus the onentrtion of VBS. Assuming tht the sturtion urve desries the inding of VBS to EGFP-vinulin, the est fit of the dt gve K d of.5 µm. representtive of 2 experiments is shown. 5