Name: generating a fractal microstructure of laminin-111 to signal to cells
Uploaded: 2020-09-28T13:00:00
Description: Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

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.

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.
<ol>
	<li>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...

<ol>
	<li>Carey, S. P., Kraning-Rush, C. M., Williams, R. M., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+control+of+invasive+tumor+cell+behavior+by+extracellular+matrix+microarchitecture.">Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture.</a> Biomaterials. 33 (16), 4157-4165 (2012).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffening+and+unfolding+of+early+deposited-fibronectin+increase+proangiogenic+factor+secretion+by+breast+cancer-associated+stromal+cells.">Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells.</a> Biomaterials. 54, 63-71 (2015).</li><li>Bruekers, S. M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin-fiber+architecture+influences+cell+spreading+and+differentiation.">Fibrin-fiber architecture influences cell spreading and differentiation.</a> Cell Adhesion &amp; Migration. 10 (5), 495-504 (2016).</li><li>Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+Activities+of+Laminins+that+Drive+Basement+Membrane+Assembly+and+Function.">Integrating Activities of Laminins that Drive Basement Membrane Assembly and Function.</a> Curr Top Membr. 76, 1-30 (2015).</li><li>Colognato, H., Winkelmann, D. A., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+Polymerization+Induces+a+Receptor-Cytoskeleton+Network.">Laminin Polymerization Induces a Receptor-Cytoskeleton Network.</a> The Journal of Cell Biology. 145 (3), 619-631 (1999).</li><li>Powell, S. K., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+laminins+and+their+cellular+receptors.">Neuronal laminins and their cellular receptors.</a> International Journal of Biochemistry &amp; Cell Biology. 29 (3), 401-414 (1997).</li><li>Li, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+a+reconstituted+basement+membrane+and+its+components+on+casein+gene+expression+and+secretion+in+mouse+mammary+epithelial+cells.">Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (1), 136-140 (1987).</li><li>Fiore, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin-111+and+the+Level+of+Nuclear+Actin+Regulate+Epithelial+Quiescence+via+Exportin-6.">Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6.</a> Cell Rep. 19 (10), 2102-2115 (2017).</li><li>Inman, J. L., Robertson, C., Mott, J. D., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development:+cell+fate+specification,+stem+cells+and+the+microenvironment.">Mammary gland development: cell fate specification, stem cells and the microenvironment.</a> Development. 142 (6), 1028-1042 (2015).</li><li>Weaver, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> Journal of Cell Biology. 137 (1), 231-245 (1997).</li><li>Furuta, S., Ren, G., Mao, J. -. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+signals+initiate+the+reciprocal+loop+that+informs+breast-specific+gene+expression+and+homeostasis+by+activating+NO,+p53+and+microRNAs.">Laminin signals initiate the reciprocal loop that informs breast-specific gene expression and homeostasis by activating NO, p53 and microRNAs.</a> eLife. 7, 26148 (2018).</li><li>Brown, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophic+phenotype+induced+in+vitro+by+antibody+blockade+of+muscle+alpha-dystroglycan-laminin+interaction.">Dystrophic phenotype induced in vitro by antibody blockade of muscle alpha-dystroglycan-laminin interaction.</a> Journal of Cell Science. 112 (2), 209 (1999).</li><li>Calof, A. L., Campanero, M. R., O'Rear, J. J., Yurchenco, P. D., Landert, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain-specific+activation+of+neuronal+migration+and+neurite+outgrowth-promoting+activities+of+laminin.">Domain-specific activation of neuronal migration and neurite outgrowth-promoting activities of laminin.</a> Neuron. 13 (1), 117-130 (1994).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin-sulfatide+binding+initiates+basement+membrane+assembly+and+enables+receptor+signaling+in+Schwann+cells+and+fibroblasts.">Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.</a> The Journal of Cell Biology. 169 (1), 179-189 (2005).</li><li>Sonnenberg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+recognition+of+different+cell-binding+fragments+of+laminin+(P1,+E3,+E8)+and+evidence+that+alpha+6+beta+1+but+not+alpha+6+beta+4+functions+as+a+major+receptor+for+fragment+E8.">Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.</a> The Journal of Cell Biology. 110 (6), 2145-2155 (1990).</li><li>Sorokin, L., Sonnenberg, A., Aumailley, M., Timpl, R., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+the+laminin+E8+cell-binding+site+by+an+integrin+possessing+the+alpha+6+subunit+is+essential+for+epithelial+polarization+in+developing+kidney+tubules.">Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.</a> The Journal of Cell Biology. 111 (3), 1265-1273 (1990).</li><li>Horejs, C. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically-active+laminin-111+fragment+that+modulates+the+epithelial-to-mesenchymal+transition+in+embryonic+stem+cells.">Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 111 (16), 5908-5913 (2014).</li><li>Hochman-Mendez, C., Cantini, M., Moratal, D., Salmeron-Sanchez, M., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fractal+Nature+for+Polymerized+Laminin.">A Fractal Nature for Polymerized Laminin.</a> PLOS ONE. 9 (10), 109388 (2014).</li><li>Kent, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microstructure+of+laminin-111+compensates+for+dystroglycan+loss+in+mammary+epithelial+cells+in+downstream+expression+of+milk+proteins.">The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins.</a> Biomaterials. 218, 119337 (2019).</li><li>Weir, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystroglycan+loss+disrupts+polarity+and+beta-casein+induction+in+mammary+epithelial+cells+by+perturbing+laminin+anchoring.">Dystroglycan loss disrupts polarity and beta-casein induction in mammary epithelial cells by perturbing laminin anchoring.</a> Journal of Cell Science. 119, 4047-4058 (2006).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Palmero, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+follicular+thyroid+cell+line+PCCL3+responds+differently+to+laminin+and+to+polylaminin,+a+polymer+of+laminin+assembled+in+acidic+pH.">The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.</a> Molecular and Cellular Endocrinology. 376 (1), 12-22 (2013).</li><li>Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylaminin+recognition+by+retinal+cells.">Polylaminin recognition by retinal cells.</a> Journal of Neuroscience Research. 92 (1), 24-34 (2014).</li><li>Leal-Lopes, C., Grazioli, G., Mares-Guia, T. R., Coelho-Sampaio, T., Sogayar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+laminin+incorporation+into+alginate-based+microcapsules+reduces+pericapsular+overgrowth+and+inflammation.">Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (10), 1912-1922 (2019).</li><li>Paccola Mesquita, F. C., Hochman-Mendez, C., Morrissey, J., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+as+a+Potent+Substrate+for+Large-Scale+Expansion+of+Human+Induced+Pluripotent+Stem+Cells+in+a+Closed+Cell+Expansion+System.">Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.</a> Stem Cells International. 2019, 9 (2019).</li><li>Walker, J. M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , 61-67 (2002).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

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...

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 &#945;1, is necessary for binding dystroglycan and integrins &#945;6&#946;1 and &#945;3&#946;115, 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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% formalin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">HT501128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin primary antibody</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Rabbit Alexafluor 488 secondary antibody</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A32731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C4901 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">Heracell 150 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge for microcentrifuge tubes</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:161px;">5418 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal Microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">LSM 710 or equivalent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">25CIR-1 or similar</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">11330107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF (Epidermal Growth Factor)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">11376454001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescently Labeled Laminin</td>
			<td style="width:121px;">Cytoskeleton Inc</td>
			<td style="width:161px;">LMN02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">VWRV0304-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">320331 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortisone</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">H0888-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">16634-50mg</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MepG Dystroglycan Knockout Cells</td>
			<td style="width:121px;">LBNL</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S8045 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normocin</td>
			<td style="width:121px;">Invivogen</td>
			<td style="width:161px;">ant-nr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium Tetroxide</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">75633</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovine Prolactin</td>
			<td style="width:121px;">Los Angeles Biomedical Research Institute</td>
			<td style="width:161px;">Ovine Pituitary Prolactin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phalloidin</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A22287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">10010023 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purified Laminin-111</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Recombinant human Nidogen-1, carrier free</td>
			<td style="width:121px;">R&#38;D systems</td>
			<td style="width:161px;">2570-ND-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Acetate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S2889 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cacodylate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile filters</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">SLGP033RS or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">252859 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-low protein binding tubes</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">76322-516, 76322-522 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure water</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">15230253 or similar</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a fractal microstructure of laminin-111 to signal to cells

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.

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...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

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

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 ...

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

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