We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.
Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.
Unlike growth factors or cytokines, extracellular matrix (ECM) proteins can assemble into structural networks. As ECM dysfunction is a key element of many disease conditions, the mechanisms by which cells sense and transduce signals from ECM composition, microstructure and biomechanics are attractive targets for therapeutic development. Growing evidence suggests that microstructure of these networks plays a major role in how these proteins signal to cells. To date, this linkage between ECM microstructure and cell function has been shown for collagen I1, fibronectin2, and fibrin3.
Laminin-111 (Ln1) is a trimeric, cross-shaped protein that is a key structural component of the extracellular matrix that contacts epithelial cells4, muscle cells5, and neural cells6. In the mammary gland, Ln1 is necessary for functional differentiation of mammary gland epithelial cells into milk producing cells 7,8,9, and Ln1 is crucial for induction of tumor reversion10,11. Ln1 and other laminins are necessary for development of cortical actin networks and sarcolemma organization in muscle12,13, and for neural cell migration and neurite outgrowth13 . Thus, understanding the mechanisms by which laminin signals to cells is an active area of research.
Ln1 contains multiple adhesive domains for a broad range of receptors including integrins, syndecans, dystroglycan, LBP-110 and LamR (Figure 1A)4,14. The E8 fragment, which contains the c-terminus globular domains of laminin α1, is necessary for binding dystroglycan and integrins α6β1 and α3β115, and it is necessary for functional differentiation of epithelial cells15,16. In contrast, the short arms show different biological activity17, meaning that availability for recognition of these different Ln1 domain after network formation could alter cell behavior.
We have recently demonstrated that Ln1 arranged into a polymeric network exhibits fractal properties (i.e., a structure that is self-similar across multiple length scales)18. Fractal networks signal differently to epithelial cells compared to Ln1, which lacks this arrangement19. This fractal network indicates a particular assembly structure, where the long arm is preferentially exposed to cells18. As a result of this different long arm display, epithelial cells undergo functional differentiation into milk producing cells with well-organized tight junctions and suppression of actin stress fibers19.
We describe 3 methods to generate Ln1 networks from short arm-short arm binding interactions, which show a high display of the Ln1 long arm: 1) by cell-free assembly with acidic buffers, 2) by co-incubation with glycoproteins, or 3) by interaction with cell surface dystroglycan. These three methods generate similar laminin microstructures through three very different mechanisms, permitting flexibility in experimental design. Previous work suggests that these three methods could represent biological robustness: in mammary gland epithelia, either cell surface dystroglycan, glycoproteins, or artificially structured laminin can induce functional differentiation19. Thus, researchers can choose between these methods based on the study subject: dystroglycan expressing cells show no sensitivity to Ln-1 microstructure, as dystroglycan in the cell membrane induces correct polymerization into a fractal network19,20. Matrigel, a mix of laminins and glycoproteins21, represents the most economical source of Ln-1, and offers the necessary microstructure to induce dystroglycan knockout mammary cells to differentiate20, but contains a complex mixture of proteins with lot to lot variability. PolyLM represents the cleanest, most reductionist system that provides this function, but at increased cost and lower efficacy to induce mammary cell differentiation compared to Matrigel19.
These three methods have a fractal network structure characteristic of diffusion limited aggregation18,19, and show altered biological activity compared to aggregated laminin in multiple experimental systems22,23,24,25. Future studies using these methods could identify the receptors and signaling necessary for epithelial differentiation and determine to restore normal cell-matrix interactions in cells in abnormal microenvironments20, such as tumors.
Laminins represent a key element of the ECM in epithelial, muscular, and neural organ systems, and have been shown in vitro to play essential roles in regulating the functional differentiation of cells. Growing evidence indicates that the structure of laminin displayed to cells regulates its signaling and resultant cell phenotype15,16, thus methods to control Ln1 microstructure should be of interest to basic biologists working in these fields, and to tissue engin…
The authors have nothing to disclose.
This work was funded by Lawrence Livermore National Lab LDRD 18-ERD-062 (to C.R.). Thanks to John Muschler for his kind gift of the DgKO and DgKI cells. Thank you to the staff at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection.
10% formalin | Sigma Aldrich | HT501128 | |
Anti-Laminin primary antibody | Sigma Aldrich | L9393 | |
Anti-Rabbit Alexafluor 488 secondary antibody | Thermo | A32731 | |
Bovine Serum Albumin | Sigma Aldrich | A9418 | |
CaCl2 | Sigma Aldrich | C4901 or similar | |
Cell Culture Incubator | Thermo | Heracell 150 or similar | |
Centrifuge for microcentrifuge tubes | Eppendorf | 5418 or similar | |
Confocal Microscope | Zeiss | LSM 710 or equivalent | |
Coverslips | Thermo | 25CIR-1 or similar | |
DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red | Thermo | 11330107 | |
EGF (Epidermal Growth Factor) | Sigma Aldrich | 11376454001 | |
Fluorescently Labeled Laminin | Cytoskeleton Inc | LMN02 | |
Gentamicin | VWR | VWRV0304-10G | |
Glutaraldehyde | Sigma Aldrich | G5882 | |
HCl | Sigma Aldrich | 320331 or similar | |
Hydrocortisone | Sigma-Aldrich | H0888-5G | |
Insulin | Sigma Aldrich | 16634-50mg | |
MepG Dystroglycan Knockout Cells | LBNL | N/A | |
NaOH | Sigma Aldrich | S8045 or similar | |
Normocin | Invivogen | ant-nr-1 | |
Osmium Tetroxide | Sigma Aldrich | 75633 | |
Ovine Prolactin | Los Angeles Biomedical Research Institute | Ovine Pituitary Prolactin | |
Phalloidin | Thermo | A22287 | |
Phosphate Buffered Saline | Thermo | 10010023 or similar | |
Purified Laminin-111 | Sigma Aldrich | L2020-1MG | |
Recombinant human Nidogen-1, carrier free | R&D systems | 2570-ND-050 | |
Sodium Acetate | Sigma Aldrich | S2889 or similar | |
Sodium Cacodylate | Sigma Aldrich | C0250 | |
Sterile filters | Millipore | SLGP033RS or similar | |
Tris | Sigma Aldrich | 252859 or similar | |
Triton X-100 | Sigma-Aldrich | X100 | |
Ultra-low protein binding tubes | VWR | 76322-516, 76322-522 or similar | |
Ultrapure water | Thermo | 15230253 or similar |