Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.

The movements of beads pulled by several kinesin-1 (conventional kinesin) motors are studied both theoretically and experimentally. While the velocity is approximately independent of the number of motors pulling the beads, the walking distance or run-length is strongly increased when more motors are involved. Run-length distributions are measured for a wide range of motor concentrations and matched to theoretically calculated distributions using only two global fit parameters. In this way, the maximal number of motors pulling the beads is estimated to vary between two and seven motors for total kinesin concentrations between 0.1 and 2.5 μg/ml or between 0.27 and 6.7 nM. In the same concentration regime, the average number of pulling motors is found to lie between 1.1 and 3.2 motors.

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.

No abstract

Conventional kinesin is a motor protein which translocates organelles from cell centre to cell periphery along specialized filamentous tracks, called microtubules. The direction of translocation is determined by microtubule polarity. This process of biological force generation can be simulated outside cells with kinesin-coated particles actively moving along immobilized microtubules. The in vitro approaches of kinesin-mediated transport described so far had the disadvantage that concerning their polarity the microtubules were randomly distributed resulting in random transport direction. The present paper demonstrates the unidirectional translocation of kinesin-coated cargoes across arrays of microtubules aligned not only in a geometrically parallel but also in an isopolar fashion. As cargo, glass, gold, and polystyrene beads with diameters between 1 and 10 µm were used. Independent of material and size, these beads were observed to move unidirectionally with average velocities of 0.3-1.0 µm s-1 over distances up to 2.2 mm. Moreover, the isopolar microtubule arrays even enabled the transport of large flat glass particles with an area of up to 24 µm×12 µm and 2-5 µm thickness which obviously contacted more than one microtubule. The controlling transport direction is considered to be an essential step for future developments of motor protein-based microdevices working in nanometre steps.