Adhesive Micropatterns to Study Intermediate Filament Function in Nuclear Positioning

Isabelle Dupin1, Julien Elric2, Sandrine Etienne‐Manneville2

1 INSERM, Centre de Recherche Cardio‐Thoracique de Bordeaux, Bordeaux, 2 Institut Pasteur, Cell polarity and migration group and CNRS URA 2582
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.7
DOI:  10.1002/0471143030.cb1307s66
Online Posting Date:  March, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The nucleus is generally found near the cell center; however its position can vary in response to extracellular or intracellular signals, leading to a polarized intracellular organization. Nuclear movement is mediated by the cytoskeleton and its associated motors. While the role of actin and microtubule cytoskeletons in nuclear positioning has been assessed in various systems, the contribution of intermediate filaments is less established due in part to the lack of tools to study intermediate filament functions. The methods described here use micropatterned substrates to impose reproducible cell shape and nucleus position. Intermediate filament organization can be perturbed using gene downregulation or upregulation; intermediate filaments can also be visualized using fluorescent intermediate filament proteins. This protocol is valuable for characterizing the role of intermediate filaments in a variety of live or fixed adherent cells. © 2015 by John Wiley & Sons, Inc.

Keywords: nucleus; polarity; cytoskeleton; micropattern; videomicroscopy

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Microcontact Printing
  • Alternate Protocol 1: Micropatterning with Deep UV Light
  • Basic Protocol 2: Cell Deposition on Micropatterns
  • Support Protocol 1: Culturing Primary Astrocytes
  • Basic Protocol 3: Quantitative Analysis of Nuclear Positioning and Perinuclear Intermediate Filaments Accumulation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Microcontact Printing

  Materials
  • Rat astrocytes (or other cells of interest; see Etienne‐Manneville, and protocol 4Support Protocol)
  • Sylgard 184 silicone elastomer kit (vinyl‐terminated polydimethylsiloxane [PDMS] plus curing agent)
  • Fibronectin solution from human plasma, 0.1% (cat. no. F0895, Sigma‐Aldrich)
  • PLL(20)‐g[3.5]‐PEG(2) (PEG‐poly(L‐lysine), SuSoS; see recipe)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Absolute ethanol
  • Acetone
  • 1 M N‐(2‐hydroxyethyl)piperazine‐N′‐2‐ethanesulfonic acid (HEPES) solution (see recipe)
  • Graphic design software (e.g., Illustrator)
  • Double‐sided tape
  • Vacuum bell
  • Petri dishes (32‐ and 100‐mm diameter)
  • 60°C incubator
  • Scalpel
  • Plastic glass and fork
  • 18‐mm diameter glass coverslips (Marienfeld GmBH)
  • Ultrasonic cleaner (Fisherbrand LEO‐50)
  • Whatman paper
  • Spin columns, Costar Spin‐X centrifuge tube filter, pore size 0.22 μm, Nylon membrane (for preparation of Texas Red dextran solution, see recipe; cat. no. CLS8169, Sigma‐Aldrich)
  • Microcentrifuge (SIGMA 1‐14 microfuge)
  • 37°C, 5% CO 2 cell culture incubator
  • Spinning disk microscope
  • Adapted device to mount coverslips for spinning disk microscope
  • Absorbing paper
  • 12‐well tissue culture plate

Alternate Protocol 1: Micropatterning with Deep UV Light

  Additional Materials (see protocol 1)
  • PLL(20)‐g[3.5]‐PEG(2) solution (PEG‐poly(L‐lysine), PLL‐g‐PEG; SuSoS; see recipe)
  • Fibronectin solution (1 mg/ml, catalog no. F1141, Sigma‐Aldrich)
  • 100 mM sodium bicarbonate (NaHCO 3) solution (pH 8.5, see recipe)
  • 70% (v/v) ethanol
  • 18‐mm diameter glass coverslips (Marienfeld GmBH)
  • Plasma cleaner, 230 V (model PDC‐002, Harrick Plasma)
  • Ultraviolet (UV) ozone oven, 185 nm (wavelength must be <200 nm) equipped with ozone catalyzer (UVO cleaner, model 342‐220, Jelight)
  • Metal block support (to hold coverslips during UV illumination; dimensions adapted to UV oven, homemade)
  • Synthetic Quartz mask with features (Toppan Photomasks)
  • Kimtech Science Precision wipes (reference no. 05511, Kimberly‐Clark Professional)
  • Wash‐N‐Dry coverslip rack (catalog no. Z688568‐1EA, Sigma‐Aldrich)

Basic Protocol 2: Cell Deposition on Micropatterns

  Materials
  • Primary rat astrocytes (see Etienne‐Manneville, and protocol 4Support Protocol)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Poly‐L‐ornithine solution (1.5 mg/ml; see recipe)
  • Bovine serum albumin (BSA)
  • 0.25% trypsin/0.02% EDTA solution (see recipe)
  • Appropriate cell culture medium (e.g., Dulbecco's modified Eagle medium [DMEM] low glucose, 2 mM L‐glutamine, supplemented with 10% fetal bovine serum [FBS], penicillin/streptomycin and fungizone [for primary astrocytes])
  • Imaging medium (e.g., HEPES, DMEM low glucose with sodium pyruvate, without red phenol and glutamine, supplemented with glutamax and 250 nM Trolox)
  • siRNAs for intermediate filaments (Table 13.7.1)
  • DNA pLifeact‐Cherry (gift from M. Piel, Institut Curie, Paris, France; Riedl et al., )
  • pVimentin‐green fluorescent protein (GFP; gift from D. Pham‐Dinh, INSERM UMR 546, Paris, France; Mignot et al., )
  • 4% paraformaldehyde (PFA) in H 2O (see recipe)
  • Methanol
  • Sodium borohydride (NaBH 4)
  • Triton X100 (Sigma‐Aldrich)
  • Prolong Gold antifade mountant with 4′,6‐diamidino‐2‐phenylindole (DAPI; Life Technologies)
  • Primary antibodies (see Table 13.7.2)
  • Secondary fluorescent antibodies
  • Hoechst and fluorescent phalloidin
  • 32‐mm and 100‐mm diameter petri dishes
  • Hemocytometer
  • Nucleofector device and nucleofector kit for cells, including cuvette holder (Lonza)
  • Adapted tweezers (to hold coverslips)
  • Coverslip holding device (Life Imaging Services)
  • Stericup filter unit (0.22‐μm pore size)
  • Spinning‐disk confocal microscope equipped with a 37°C heating system for the stage
  • Conventional epifluorescence microscope
  • Centrifuge (for the cells and plates; Beckman Coulter Allegra X‐22R)
  • Falcon tubes (Fisher Scientific)
  • 12‐well tissue culture plate
  • 18‐mm diameter glass coverslips (Marienfeld GmBH)
Table 3.7.1   MaterialssiRNA SequencesAntibodies Used for Immunofluorescence

Name and accession number Position inside gene a Positive‐sense sequence Expression decrease b
GFAP NM_017009 siRNA: 757 GAGUGGUAUCGGUCCAAGUdTdT 89%
Vimentin NM_031140 siRNA: 781 UGAAGAAGCUGCACGAUGAdTdT 97%
Nestin NM_012987 siRNA: 780 GUUCCAGCUGGCUGUGGAAdTdT 90%
Antibody directed against Host species Monoclonal (M) or polyclonal (P) Supplier Suitable fixation Dilution
Pan‐cadherin Mouse M Sigma‐Aldrich PFA/methanol 1/100
N‐cadherin Rabbit P Abcam PFA/methanol 1/100
E‐cadherin Rabbit P Santa Cruz PFA/methanol 1/50
GFAP Goat P Santa Cruz PFA 1/100
GFP Mouse M Roche PFA 1/100
Nestin Mouse M Chemicon PFA 1/100
α‐Tubulin Rat M Serotec Methanol 1/100
Vimentin Goat P Santa Cruz PFA 1/100

 aPosition in the gene: indicates the position in the gene relative to the beginning of the sequence (given by the accession number)
 bThe decrease of protein expression is calculated by measuring the average intensity of the bands on western blot. Background noise measurement is subtracted from each average intensity measurement. This value is then normalized relative to the expression of a control protein (β‐catenin or tubulin) not targeted by the siRNA.
Table 3.7.2   MaterialssiRNA SequencesAntibodies Used for Immunofluorescence

Name and accession number Position inside gene a Positive‐sense sequence Expression decrease b
GFAP NM_017009 siRNA: 757 GAGUGGUAUCGGUCCAAGUdTdT 89%
Vimentin NM_031140 siRNA: 781 UGAAGAAGCUGCACGAUGAdTdT 97%
Nestin NM_012987 siRNA: 780 GUUCCAGCUGGCUGUGGAAdTdT 90%
Antibody directed against Host species Monoclonal (M) or polyclonal (P) Supplier Suitable fixation Dilution
Pan‐cadherin Mouse M Sigma‐Aldrich PFA/methanol 1/100
N‐cadherin Rabbit P Abcam PFA/methanol 1/100
E‐cadherin Rabbit P Santa Cruz PFA/methanol 1/50
GFAP Goat P Santa Cruz PFA 1/100
GFP Mouse M Roche PFA 1/100
Nestin Mouse M Chemicon PFA 1/100
α‐Tubulin Rat M Serotec Methanol 1/100
Vimentin Goat P Santa Cruz PFA 1/100

Support Protocol 1: Culturing Primary Astrocytes

  Materials
  • 18‐day pregnant Sprague‐Dawley rat
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 0.4% glucose solution (see recipe)
  • Dulbecco's modified Eagle medium (DMEM) low glucose, 2 mM L‐glutamine, supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and fungizone (culture medium)
  • 1.5 mg/ml poly‐L‐ornithine solution (see recipe)
  • 32‐, 60‐, and 100‐mm diameter petri dishes
  • Dissection instruments (scissors, curved scissors, tweezers, fine tweezers)
  • P1000 pipet
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Basic Protocol 3: Quantitative Analysis of Nuclear Positioning and Perinuclear Intermediate Filaments Accumulation

  Materials
  • Image analysis software (e.g., ImagJ)
  • Calculation software (e.g., Excel)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Azioune, A., Storch, M., Bornens, M., Thery, M., and Piel, M. 2009. Simple and rapid process for single cell micro‐patterning. Lab. Chip. 9:1640‐1642.
  Azioune, A., Carpi, N., Tseng, Q., Thery, M., and Piel, M. 2010. Protein micropatterns: A direct printing protocol using deep UVs. Methods. Cell Biol. 97:133‐146.
  Badyal, S.K., Basran, J., Bhanji, N., Kim, J.H., Chavda, A.P., Jung, H.S., Craig, R., Elliott, P.R., Irvine, A.F., Barsukov, I.L., Kriajevska, M., and Bagshaw, C.R. 2011. Mechanism of the Ca2+‐dependent interaction between S100A4 and tail fragments of nonmuscle myosin heavy chain IIA. J. Mol. Biol. 405:1004‐1026.
  Burakov, A.V. and Nadezhdina, E.S. 2013. Association of nucleus and centrosome: Magnet or velcro? Cell Biol. Int. 37:95‐104.
  Donovan, J. and Brown, P. 2005. Euthanasia. Curr. Protoc. Neurosci. 33:A.4H.1‐A.4H.4.
  Dupin, I. and Etienne‐Manneville, S. 2011. Nuclear positioning: Mechanisms and functions. Int. J. Biochem. Cell B. 43:1698‐1707.
  Dupin, I., Sakamoto, Y., and Etienne‐Manneville, S. 2011. Cytoplasmic intermediate filaments mediate actin‐driven positioning of the nucleus. J. Cell Sci. 124:865‐872.
  Etienne‐Manneville, S. 2006. In vitro assay of primary astrocyte migration as a tool to study Rho GTPase function in cell polarization. Methods Enzymol. 406:565‐578.
  Gomes, E.R., Jani, S., and Gundersen, G.G. 2005. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121:451‐463.
  Gundersen, G.G. and Worman, H.J. 2013. Nuclear positioning. Cell 152:1376‐1389.
  Henikoff, S. 2011. Summary: The nucleus–a close‐knit community of dynamic structures. Cold Spring Harb. Symp. Quant. Biol. 75:607‐615.
  Isermann, P. and Lammerding, J. 2013. Nuclear mechanics and mechanotransduction in health and disease. Curr. Biol. 23:R1113‐R1121.
  Kutscheidt, S., Zhu, R., Antoku, S., Luxton, G.W., Stagljar, I., Fackler, O.T., and Gundersen, G.G. 2014. FHOD1 interaction with nesprin‐2G mediates TAN line formation and nuclear movement. Nat. Cell Biol. 16:708‐715.
  Lombardi, M.L., Jaalouk, D.E., Shanahan, C.M., Burke, B., Roux, K.J., and Lammerding, J. 2011. The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J. Biol. Chem. 286:26743‐26753.
  Mignot, C., Delarasse, C., Escaich, S., Della Gaspera, B., Noe, E., Colucci‐Guyon, E., Babinet, C., Pekny, M., Vicart, P., Boespflug‐Tanguy, O., Dautigny, A., Rodriguez, D., and Pham‐Dinh, D. 2007. Dynamics of mutated GFAP aggregates revealed by real‐time imaging of an astrocyte model of Alexander disease. Exp. Cell Res. 313:2766‐2779.
  Quist, A.P. and Oscarsson, S. 2010. Micropatterned surfaces: Techniques and applications in cell biology. Expert Opin. Drug Discov. 5:569‐581.
  Riedl, J., Crevenna, A.H., Kessenbrock, K., Yu, J.H., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T.A., Werb, Z., Sixt, M., and Wedlich‐Soldner, R. 2008. Lifeact: A versatile marker to visualize F‐actin. Nat. Methods. 5:605‐607.
  Samora, C.P., Mogessie, B., Conway, L., Ross, J.L., Straube, A., and McAinsh, A.D. 2011. MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis. Nat. Cell Biol. 13:1040‐1050.
  Singhvi, R., Kumar, A., Lopez, G.P., Stephanopoulos, G.N., Wang, D.I., Whitesides, G.M., and Ingber, D.E. 1994. Engineering cell shape and function. Science 264:696‐698.
  Webb, D.J., Donais, K., Whitmore, L.A., Thomas, S.M., Turner, C.E., Parsons, J.T., and Horwitz, A.F. 2004. FAK‐Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6:154‐161.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library