Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Summary

We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

Abstract

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.

Introduction

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 reversion^10,11. Ln1 and other laminins are necessary for development of cortical actin networks and sarcolemma organization in muscle^12,13, 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 cells^15,16. 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 network^19,20. 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 aggregation^18,19, and show altered biological activity compared to aggregated laminin in multiple experimental systems^22,23,24,25. 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.

Protocol

1. Thaw and aliquot laminin-111 NOTE: Purified laminin-111 is available commercially (typically purified from EHS matrix sold as Matrigel), but not all sources provide intact, nondegraded protein. Confirm that Ln1 is not degraded by sourcing Ln1 and running size exclusion chromatography26 or polyacrylamide gel electrophoresis26. Denatured Ln1 should have bands at 337 kDa, 197 kDa and 177 kDa, and native Ln1 should have a single band a…

Representative Results

Laminin-111 is a trimeric protein with three self-assembling domains at the end of its short arms (Figure 1A). When treated suitably, the short arms can polymerize into a hexagonal lattice (Figure 1B,1C), which displays diffusion-limited aggregate fractal properties at larger spatial scales (Figure 2). Laminin incubated in calcium-containing neutral-pH buffers forms small aggregates visualized with an anti-Ln1 antib…

Discussion

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 phenotype^15,16, thus methods to control Ln1 microstructure should be of interest to basic biologists working in these fields, and to tissue engin…

Materials

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