Tamra se

Tubulin was purified from porcine brain using a high-molarity PIPES buffer (HMPB) (1 M PIPES, 20 mM EGTA, 10 mM MgCl₂; pH adjusted to 6.8 using KOH). HMPB and BRB80 buffer were prepared using PIPES from Sigma, and the pH was adjusted using KOH50 (link). GFP-fused kinesin-1 consisting of the first 560 amino acids of human kinesin-1 (GFP-kinesin) was prepared by partially modifying previously reported expression and purification methods51 (link). Rhodamine-labelled tubulin was prepared using 5/6-carboxy-tetramethyl-rhodamine succinimidyl ester (TAMRA-SE; Invitrogen) according to the standard techniques52 (link). Rhodamine-labelled tubulin was obtained by chemical crosslinking, and the labelling ratio was 1.0, as determined by measuring the absorbance of the protein at 280 nm and that of tetramethyl-rhodamine at 555 nm.

The artificial muscle specimen, composed of hierarchically ordered microtubule asters, was prepared as reported previously [4 (link)]. Short microtubule seeds were polymerized from tubulin dimers labelled with fluorescent dye Alexa 488. The seed microtubules were elongated through polymerization of azide-conjugated tubulins and tubulin dimers that were labelled with TAMRA dye. Single strand DNA with a predesigned sequence was conjugated to the elongated part of the microtubules through a click reaction. According to the standard procedure as reported in literature [9 (link)] rhodamine or Alexa488-labeled tubulin was prepared using 5/6-carboxytetramethylrhodamine succinimidyl ester (TAMRA-SE; Invitrogen, Carlsbad, CA, USA) or Alexa488, respectively, with the standard technique, and the labeling ratio was 1.0 in each case. Azide-labeled tubulin was prepared using N3-PEG4-NHS, following the protocol of labeling tubulin with fluorescent dye. After preparation, the DNA-conjugated microtubules were mixed with DNA origami to form asters. Upon addition of kinesin multimer linkers created with streptavidin molecules binding four kinesin dimers, and with the addition of ATP, the filament mesh structure contracted like muscle. We further examined this contraction scheme in our model calculation described below.

Rhodamine-labelled tubulin was prepared using 5/6-carboxy-tetramethyl-rhodamine succinimidyl ester (TAMRA-SE; Invitrogen) according to the standard technique35 (link) and the labeling ratio was 1.0 as determined by measuring the absorbance of the protein at 280 nm and that of tetramethyl-rhodamine at 555 nm. Rhodamine-labelled MTs were obtained by polymerizing a mixture of rhodamine tubulin (RT) and non-labelled tubulin (WT) at 37 °C (RT: WT = 4:1; final tubulin concentration = 55.6 μM). The solution containing the MTs was then diluted with motility buffer (80 mM PIPES, 1 mM EGTA, 2 mM MgCl₂, 0.5 mg mL⁻¹ casein, 1 mM DTT, 10 μM paclitaxel; pH 6.8).

To perform staining, the medium was aspirated from the dishes and the wells were washed (×2) with PBS. The wells were fixed with 4% PFA (Thermo Fisher Scientific) for 15 min and washed with PBS (x3, 5 min each). The collagen fibres were stained with 50 μM 5(6)-carboxytetramethylrhodamine succinimidyl ester (TAMRA-SE, Invitrogen) at 4 ᵒC overnight and rinsed 5 times with PBS.

White Leghorn chicken (Gallus gallus domesticus) eggs (n = 36 analysed) were incubated at 38°C on stationary shelves until stage 18–19 [36] (link). The outer face of the looping heart tube was tattooed with a 1.25 mg/ml red fluorescent mix of 1,1′-dioctade dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate (DiI, Invitrogen, D-282) and 5-carboxytetramethylrhodamine, succinimidyl ester (TAMRA SE, Invitrogen, C2211) as described [36] (link). Localized microinjection, using an ultrathin glass needle (inner diameter 0.01 mm) and a Narishige IM-300 microinjector, of a minimal fluorophore volume at the anterior surface near the inner curvature resulted in labelling mostly myocardial but also epicardial cells. After microinjection, the eggs were re-incubated to allow further development to stages 24–35.