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.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

Laminin is an essential part of the DCM, the extracellular matrix, especially Laminin-111 in the context of some cell types. Evidence suggests that the microstructure of laminin informs signaling cells. Polymerized laminin-1 exhibit a fractal microstructure showing robust biological activity and, as compared to conventional aggregated laminin-1.Poly laminin's fractal network indicates that an assembly structure conductive to the functional differentiation of epithelial cells as well as other cell types. This is why researchers can choose between three different methods for assembling this biologically robust laminin polymer depending on their study subject. Currently, acute local injections of poly laminin produced under pH acidification has been shown to be effective in regenerating human spinal cord injury in large animals and human clinical studies.Basic studies of poly laminin in heart failure and breast cancer are ongoing. Future studies using these methods to polymerize laminin-1 into fractal networks could identify the receptors and signaling necessary for epithelial as other cell types differentiation and investigate cell matrix interaction in both normal and abnormal microenvironments Thaw laminin-111 on ice in a refrigerator overnight. Then, use low protein binding tips to aliquot it into low protein binding micro centrifuge tubes.Freeze aliquots and store them at 80 degrees Celsius. To make the polyLM polymerization buffer, weigh out and mix one millimolar calcium chloride and 20 millimolar sodium acetate in ultrapure water. Adjust the pH to four with hydrochloric or acetic acid, then filter the buffer through a 0.2 micrometer filter and store it at four degrees Celsius.Add the polyLM polymerization buffer to thawed laminin aliquots for a final concentration of 100 micrograms per milliliter and mix the solution by pipetting. Pipette the diluted laminin on glass cover slips and incubate them at 37 degrees Celsius for 30 minutes. Seed dystroglycan knock-out and dystroglycan knock-in cells for laminin stimulation at 10, 500 cells per centimeter squared in well plates or cover slip bottom dishes.After removing the supernatant, resuspend the laminin in an appropriate volume of DMEM F12 supplemented with three microgram per milliliter prolactin, 2.8 micromolar hydrocortisone, and five microgram per milliliter insulin. Immediately, add the laminin to the seeded cells to stimulate them. Seed DG knock-in cells at 5, 000 cells per centimeter squared and DG knock-out cells at 8, 000 cells per centimeter squared and culture them at 37 degrees Celsius in a humidified incubator, changing the media every two to three days and passaging them every four to five days.Count the cells with a hemocytometer or automated cell counter to ensure appropriate growth rates. Remove media from the cells and add the laminin. Culture the cells for two days before fixing them with 10%formalin.Use a laser scanning microscope with immersion objectives to image the laminin structure. Then analyze for fractal properties using box counting dimension algorithms available in MATLAB Toolboxes or in Image J.These methods plot the number of boxes containing laminin-1 compared to the size of the boxes on a log log plot. The slope of the relationship between these parameters is the box counting dimension.Nonfractal geometries will have a dimension of zero, one, or two while fractal geometries have a non integer dimension. Laminin-111 is a trimetric protein with three self assembling domains at the end of its short arms. When treated suitably, the short arms can polymerize into a hexagonal lattice, which displays diffusion limited aggregate fractal properties at larger spatial scales.Laminin incubated in calcium containing neutral pH buffers form small aggregates, which have a box counting fractal dimension of approximately two. In contrast, laminin-1 incubated in calcium containing pH four buffer, or in neutral buffer with nidogen-1 form space filling lacey networks with a fractal dimension of 1.7. Laminin-1, cultured with dystroglycan expressing cells form similar lacey networks.When pH seven laminin and polyLM are imaged with SEM, the differences in microstructure become more apparent. At low resolution, polyLM networks appear spread out compared to pH seven laminin. At high resolution, pH seven laminin aggregates are apparent, whereas a spread hexagonal lattice can be seen in polyLM.Following this procedure, it's possible to evaluate the fractal properties of the polymers. It is important to guarantee the production of the biomemetic polymer of poly laminin. Poly laminin and pH seven laminin plus nidogen present fractional dimension.However, pH seven laminin without nidogen does not. Since poly laminin is biomemetic, the use of this fractal polymer of laminin will allow researchers to reproduce in vivo environment in an in vitro setting.

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

Research

JoVE Journal

Bioengineering

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

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

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

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How &quot;Molecular Surgery&quot; of the Backbone Affects Folding and Stability

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...