Spontaneous helical structure formation in laminin nanofibers

Abstract

Laminin is a cross-shaped heterotrimer composed of three polypeptides chains that assembles into an insoluble extracellular matrix (ECM) network as part of the basement membrane, serving a vital role in many processes such as embryonic development, differentiation, and muscle and nerve regeneration. Here we engineered monodisperse laminin nanofibers using a surface-initiated assembly technique in order to investigate how changes in protein composition affect formation and structure of the network. Specifically, we compared laminin 111 with varying degrees of purity and with and without entactin to determine whether these changes alter biophysical properties. All the laminin types were reproducibly patterned as 200 μm long, 20 μm wide nanofibers that were successfuly released during surface-initiated assembly into solution. All nanofibers contracted upon release, and while initial lengths were identical, lengths of released fibers depended on the laminin type. Uniquely, the laminin 111 at high purity (>95%) and without entactin spontaneouly formed helical nanofibers at greater than 90%. Atomic force microscopy revealed that the nanofiber contraction was associated with a change in nanostructure from fibrillar to nodular, suggestive of refolding of laminin molecules into a globular-like conformation. Further, for the high purity laminin that formed helices, the density of the laminin at the edges of the nanofiber was higher than in the middle, providing a possible origin for the differential pre-stress driving the helix formation. Together, these results show that variation in the purity of laminin 111 and presence of entactin can have significant impact on the biophysical properties of the assembled protein networks. This highlights the fact that our understanding of protein assembly and function is still incomplete and that cell-free, in vitro assays can provide unique insights into the ECM.

Stretch-dependent changes in molecular conformation in fibronectin nanofibers

“…22 Further, using laminin nanofibers, we have shown that differences in microscale mechanical behavior can be correlated directly to differences in nanoscale molecular structure of the constituent molecules. 23 In the current study, we combined uniaxial tensile deformation together with AFM imaging to directly study changes in nanoscale structure during stretch of FN nanofibers. To do this we developed a method to label the FN nanofibers with FN-based fiducial marks to accurately measure extension during uniaxial stretching, by using an adaptation of our previously published patterning-on-topography (PoT) technique.…”

Engineered Basement Membranes for Regenerating the Corneal Endothelium

“…The SIA process is able to create thin layers of dense ECM protein (Figure 1) by first partially unfolding ECM proteins in solution onto a PDMS stamp through hydrophobic interactions, and then transferring the ECM proteins in the partially unfolded state to a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) surface through microcontact printing, as previously described. [20, 23] The partially unfolded ECM proteins are then assembled in to a dense, free-standing insoluble matrix when the PIPAAm swells during the thermally-trigged dissolution process, by first hydrating the PIPAAm at 40 °C in PBS and then decreasing below the lower critical solution temperature of ~32 °C. [20, 23] We found that the gelatin carrier was critical to achieve reliable transfer of the dense ECM sheet of COL4 or COL4+LAM to the COL1 gels, as direct transfer from PIPAAm to COL1 was inconsistent due small thermal fluctuations causing premature PIPAAm dissolution.…”

“…[20, 23] The partially unfolded ECM proteins are then assembled in to a dense, free-standing insoluble matrix when the PIPAAm swells during the thermally-trigged dissolution process, by first hydrating the PIPAAm at 40 °C in PBS and then decreasing below the lower critical solution temperature of ~32 °C. [20, 23] We found that the gelatin carrier was critical to achieve reliable transfer of the dense ECM sheet of COL4 or COL4+LAM to the COL1 gels, as direct transfer from PIPAAm to COL1 was inconsistent due small thermal fluctuations causing premature PIPAAm dissolution. To confirm the laminar structure of the EBM fabricated via SIA, we used multiphoton imaging to visualize the fluorescently labeled COL4 as well as the COL1 by second harmonic generation.…”

“…In comparison, AFM of the COL4+LAM on PIPAAm at a 10 μm scan size revealed a dense layer of protein (Figure 3b), that in previous studies was determined to be 5–10 nm thick. [20, 23] At a 1 μm scan size, the COL4+LAM on PIPAAm (Figure 3e) had a mesh-like structure resembling that reported for native basement membrane, specifically the structure of Descemet’s membrane from SEM data published by Abrams et al [24] The EBM (Figure 3c) surface structure was almost identical to that of COL1 at both the 10- and 1 μm scan sizes, appearing as isotropic fibers with the banding of the underlying COL1 fibrils still visible (Figure 3f). Importantly, the surface topography due to the underlying COL1 dominated the surface structure and obscured the mesh-like structure of the COL4+LAM.…”

“…Utilizing surface initiated assembly (SIA) techniques [20] , we have fabricated an engineered basement membrane (EBM) composed of a layer of basement membrane ECM proteins that is approximately 5–10 nm thick, supported by a compressed COL1 gel that is approximately 10 μm thick (Figure 1). The dense ECM top layer of the EBM is designed to recapitulate the structure and composition of Descemet’s membrane, which in vivo consists predominantly of collagen type IV (COL4), collagen type VIII and laminin (LAM) and other ECM components in lesser amounts.…”