Abstract
Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.
Highlights
Laminin is the major molecular component of the basement membrane, a specialized type of extracellular matrix characterized by a flat sheet-like geometry
In the three-dimensional space laminin has the shape of a three-leafed clover, in which the leaves correspond to the three short arms while the stem corresponds to the long arm
It was demonstrated that polyLM, but not the laminin protein diluted in neutral buffer, promoted axonal regeneration and functional recovery after spinal cord injury in rats [10]
Summary
Laminin is the major molecular component of the basement membrane, a specialized type of extracellular matrix characterized by a flat sheet-like geometry. Using negative staining followed by transmission electron microscopy it was possible to characterize it as a regular polygonal network displaying the same features of the natural laminin networks assembled by living cells [11,12] In such polymers the unit polygon was a hexagon of approximately 30 nm of side, which well corresponded to the size of each short arm in the laminin molecule. We showed that polyLM presented a fractal dimension, which increased its complexity upon accumulation of the polymer on a flat substrate These findings may have important implications as they can provide an intrinsic molecular basis for the fractal-like organization of the basement membranes present at the neurogenic niches in the adult central nervous system
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