Figure 4. Fibrinogen anchors facilitate... | Rockefeller University Press

Figure 4.

$Fibrinogen anchors facilitate micropatterning of active motors.(A–H) Biotinylated-Kinesin1 motors (Kin1-Biotin) were micropatterned on PLL-PEG–coated glass using LIMAP either directly (A–D) or indirectly through a fibrinogen-biotin-ATTO490LS::NeutrAvidin sandwich (E–H; see Materials and methods). After washing and quenching, GMPCPP-stabilized fluorescent microtubules (MTs) were added in the presence of ATP and their motion observed by TIRFM. Dashed purple lines delineate the kinesin micropattern as imaged either through post-labeling of Kin1-Biotin by streptavidin-Alexa647 for direct micropatterning (A–D) or by fibrinogen-biotin-ATTO490LS fluorescence for the indirect micropatterning (E–H). (I) Quantification of the proportion of motile MTs in all conditions reveals that MTs outside the micropattern are immobile, in contrast to MTs landing inside the micropattern (n = 14/34/47/112, respectively). (J and K) Quantification of the speed (J; mean ± SEM) and fraction of time spent moving processively (K; mean ± SEM) reveals that MTs move faster and with fewer pauses on fibrinogen-biotin–mediated Kin1-Biotin micropatterns (directly micropatterned, n = 7; sandwich, n = 25). This suggests that indirect micropatterning of Kin1-Biotin through fibrinogen-biotin ensures high activity of the motor on the micropattern. Statistics in J and K were performed using unpaired t tests. n, number of MTs. Scale bars, 10 µm (B, D, F, and H) and 10 µm/1 min (C and G). t, time.$

Fibrinogen anchors facilitate micropatterning of active motors. (A–H) Biotinylated-Kinesin1 motors (Kin1-Biotin) were micropatterned on PLL-PEG–coated glass using LIMAP either directly (A–D) or indirectly through a fibrinogen-biotin-ATTO490LS::NeutrAvidin sandwich (E–H; see Materials and methods). After washing and quenching, GMPCPP-stabilized fluorescent microtubules (MTs) were added in the presence of ATP and their motion observed by TIRFM. Dashed purple lines delineate the kinesin micropattern as imaged either through post-labeling of Kin1-Biotin by streptavidin-Alexa647 for direct micropatterning (A–D) or by fibrinogen-biotin-ATTO490LS fluorescence for the indirect micropatterning (E–H). (I) Quantification of the proportion of motile MTs in all conditions reveals that MTs outside the micropattern are immobile, in contrast to MTs landing inside the micropattern (n = 14/34/47/112, respectively). (J and K) Quantification of the speed (J; mean ± SEM) and fraction of time spent moving processively (K; mean ± SEM) reveals that MTs move faster and with fewer pauses on fibrinogen-biotin–mediated Kin1-Biotin micropatterns (directly micropatterned, n = 7; sandwich, n = 25). This suggests that indirect micropatterning of Kin1-Biotin through fibrinogen-biotin ensures high activity of the motor on the micropattern. Statistics in J and K were performed using unpaired t tests. n, number of MTs. Scale bars, 10 µm (B, D, F, and H) and 10 µm/1 min (C and G). t, time.

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