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. Author manuscript; available in PMC: 2015 Jul 30.
Published in final edited form as: J Neurosci Methods. 2014 May 29;232:181–188. doi: 10.1016/j.jneumeth.2014.05.027

Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia

Flavia C Nery 1,#,#, Nadia A Atai 1,2,#, Cintia C da Hora 1,#, Edward Y Kim 3, Jasmin Hettich 1, Thorsten R Mempel 3, Xandra O Breakefield 1, Daniel Irimia 4
PMCID: PMC4120252  NIHMSID: NIHMS600495  PMID: 24880044

Abstract

Background

Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis.

New Method

The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease - DYT1 dystonia, have decreased migration properties when compared to control cells.

Results

We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement.

Comparison with Existing Method

The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells.

Conclusion

We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment.

Keywords: Microfluidic device, Cell polarization, Migration, Dystonia, DYT1, torsinA

1. Introduction

Dystonia is the third most common neurological movement disorder in humans. Dystonia is a complex syndrome characterized by motor dysfunction in a number of neurological diseases (for review see Albanese and Lalli, 2012). Dystonia is clinically diagnosed with physical symptoms such as involuntary twisting movements and abnormal postures (Fahn, 1984; Fung et al., 2013; Albanese et al., 2013). Fourteen genes have been implicated in 25 forms of hereditary dystonia allowing for mutational testing in diagnosis and genetic counseling (Lohmann and Klein, 2013). DYT1 dystonia, also known as early onset generalized torsion dystonia, is an autosomal-dominant form of dystonia with low penetrance with most cases caused by a common mutation - a GAG deletion in the coding region of the TOR1A gene that encodes torsinA (Bressman et al., 2002). Mutant torsinA, tors n ΔE appears to act in a dominant-negative manner to suppress wild-type activity, which supports functions of the endoplasmic reticulum (ER) and nuclear envelope (NE) (Hewett et al., 2007; Nery et al., 2008; Nery et al., 2011; Atai et al., 2012). TorsinA participates in a number of cellular functions, including migration of cells through a role in nuclear polarization (Nery et al., 2008), egress of viral and large ribonucleoprotein particles out of the NE (Maric et al., 2011; Jokhi et al., 2013), and protection from cellular stress (Nery et al., 2011; Bragg et al., 2011; Chen et al., 2010; Cao et al., 2010).

Cell migration is an evolutionarily conserved mechanism that underlies the development and functioning of uni- and multicellular organisms and takes place in normal and pathogenic processes, including various events of embryogenesis, wound healing, immune responses, cancer metastases and angiogenesis (Kurosaka and Kashina, 2008). Functionally torsinAΔE is believed to reduce activity of wild-type torsinA thereby weakening the connection between the cytoskeleton and the outer nuclear membrane and the contiguous ER membrane (Nery et al., 2008; Atai et al., 2012). The relationship between deficient cell migration and the abnormalities in synaptic plasticity found in dystonia remains to be elucidated (Albanese and Lalli, 2012; Quartarone and Pisani, 2011). The current study focuses on quantitation of changes in cell migration in DYT1 patient fibroblasts as a model for delayed migration documented for neurons in DYT1 knock-out embryos (McCarthy et al., 2012). During brain development torsinA is highly expressed in dopaminergic neurons in the central nervous system located in the substantia nigra, as well as in neurons in the striatum, cerebral cortex, thalamus, hippocampus, cerebellum, midbrain, pons and spinal cord (Rostasy et al., 2003; Augood et al., 1998, 1999, 2000; Vasudevan et al., 2006).

Microfluidic platforms are emerging to study cell migration with great spatial and temporal resolution, for precise measurements of velocity, directionality, and persistence. These tools have allowed monitoring of the vectorial movement of individual neutrophils around obstacles (Ambravaneswaran et al., 2010), cancer cells in conditions of three-dimensional confinement in linear channels (Irimia and Toner, 2009), and microglia in the presence of amyloid beta within channels (Cho et al., 2013). The unprecedented precision of speed, directionality, and persistence measurements enabled by these tools provided the support for unexpected findings regarding the alterations of neutrophil migration after burn injuries (Butler et al., 2010), the role of self-generated gradients during epithelial cell migration through mazes (Scherber et al., 2012), and the contribution of asymmetric location of mitochondria in front of the nucleus to the fast and persistent migration of cancer cells (Desai et al., 2013).

The limitations in developing neuronal models have led scientists to examine the role of proteins involved in human neurologic diseases in non-neuronal model systems (Falkenburger and Schulz, 2006). The published literature indicates this approach is not only viable, but has proven very successful, providing very useful and informative results (Ferraiuolo et al., 2013; Burbulla and Krüger, 2012; Connolli, 1998). Recently, there has been increased interest in the use of patient-derived fibroblasts, as induced pluripotent stem cells can be derived from them and studied directly or after differentiation into neurons and glia for studying neurological diseases (Koch et al., 2011; Qiang et al., 2011; Yu et al., 2007; Gibbs and Singleton, 2006). In this study we used primary skin fibroblasts from DYT1 patients and healthy controls to monitor variations in cell movement relevant to neuronal migration. We found delayed rates of migration, reduced polarization of the nucleus in migrating cells, and abnormal orientation of nuclei and Golgi in migrating DYT1, as compared to control cells using different culture assays. For the first time we report a single cell vectorial movement assay in microfluidic chambers that allows real-time evaluation of migration dynamics.

2. Materials and methods

2.1. Cell culture

The following cell lines were used, human control fibroblast lines: HF6, HF19, GM02131, GM02912B, GM0024, HF77, HF71, and DYT1 fibroblast lines: HF49, GM02551, GM02306, GM03208 and GM03211. HF lines were obtained by skin biopsy generated in our laboratory under IRB approved guidelines (Breakefield et al., 1981); GM lines were purchased from Cor l (C m n NJ). Fibroblasts were grown in Dulbecco's Modified Eagle Medium (Gibco, Rockville, MD; Invitrogen Life Technologies; Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Invitrogen Life Technologies). To simplify the text, we have used the last two/four numbers of each line of fibroblasts from Coriel in the text and figures.

2.2. Oris cell migration assay

In some experiments cell migration was analyzed using the Oris Cell Migration Assay (Platypus Technologies, Madison, WI) with some modifications. The cell-seeding stopper was inserted into wells of a 96-w ll pl t n w ll ottoms w r o t w th 10 μ /ml ron t n (Invitrogen). The fibroblasts were diluted to 2.5 x 105 lls/ml n 100 μl of the cell suspension were seeded per well with a stopper inserted to allow seeding only in the outer annular regions of the wells. After appropriate cell attachment was achieved (24 hours), the culture insert/stopper was removed and the wells were filled with cell-free medium. Removal of the stopper resulted in opening of a round, unseeded region into which the cells migrated. Cells were examined microscopically to monitor progression of migration into the cell-free zone 72 hours after stoppers were removed. Cells were fixed in 4% formaldehyde in 1X phosphate buffered saline (PBS) and stained by immunocytochemistry for an ER protein, protein disulfide isomerase (PDI) for fluorescence quantification of cells in this zone using a confocal microscope. The images were captured and the data analyzed in pre-migration (t = 0 hour) and post-migration (t = 72 hours) wells. The average fluorescence signal from the detection zones (unseeded region or cell-free zone created by insertion of the Or s™ stopper) was calculated as the mean ± S.D. for each condition.

2.3. Monolayer wounding and determination of nucleus and centrosome position

Human fibroblasts were cultured, as described above, and plated in semiconfluent monolayer. Cells were starved in serum-free medium for 2 days, because serum may contain up to 10 μM lysophosphatidic acid (LPA), a cell polarization stimulator. On the third day, three linear wounds were scratched in each well of cells with a p200 pipet tip. Then the wells were washed twice with serum-free Dulbecco's Modified Eagle's Medium (DMEM). After a wound area was created and washed, the culture medium was replaced with fresh serum-free medium containing 10 μM LPA (Sigma-Aldrich, St. Louis, MO), as described (Nery et al., 2008). Three hours later, the cells were fixed in 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature followed by methanol at -20°C for 5 minutes. Cells were stained for the centrosome marker, pericentrin (PRB-432C, from Covance, Princeton, NJ, 1:1000), nuclei (DAPI, Sigma-Aldrich 0.1%) microtubules (α-tubulin, Sigma-Aldrich, 1:1000), as described (Nery et al., 2008). Centrosome orientation relative to the position of the nucleus and leading edge of the plasma membrane was determined, as described (Gomes et al., 2005; Palazzo et al., 2001). Briefly, images were pseudocolored and analyzed with Image-J software scoring >80 cells per group. Centrosome orientation was defined by localization in the sector (~1/3 of cell) between the nucleus and leading edge (Gomes et al., 2005). Random orientation would be expected to be ~33%.

2.4. Microfluidic device manufacturing and setup

Microfluidic devices were fabricated using standard micro-fabrication technologies. Briefly, two layers of SU-8 photoresist (Microchem, Newton, MA) one 10 μm th n n on 50 μm thick were photo-patterned on a silicon wafer using standard microfabrication technologies. The thin and thick layers were aligned with 1 micron precision by using matching photolithographic features in the two layers. The design of the devices consists of four arrays of 80 parallel microchannels 10 μm width 10 μm height and 600 μm length which are distributed around a circular well of 2 mm diameter and 50 μm height. The silicon wafer with microscale SU-8 features was then used to cast polydimethyl siloxane (PDMS, Dow Corning Midland, MI) in a 2 mm thick layer. After baking for 8 hours at 65°C, the PDMS layer was peeled off the wafer, punched with a 1.5 mm puncher (Harris Uni-Core, Ted Pella, Reading, CA) to define the cell loading chamber, and whole devices punched out using a 5 mm puncher (Ted Pella). The “donut” shaped devices were then xposed to oxygen plasma and bonded on glass-bottom multiwell plates (MatTek Co., Ashland, MA). To promote adhesion of fibroblasts inside the channels, the device was primed with 10 μ/ml fibronectin for about 10 minutes. After incubation, the device was washed with PBS (Fisher Scientific, Pittsburgh, PA) and primed with DMEM media.

2.5. Microscopy and image analysis

Ten microliters of fibroblasts (3000 to 10000 cells) were injected inside of the cell loading chamber of each microfluidic device. The migration of the cells from the chamber into the channels was recorded using a Nikon TiE microscope operating in epifluorescence mode and equipped with a culture incubation chamber. All fluorescence images were acquired with 12-bit resolution using a cooled-CCD camera (QImaging, Surrey, BC, Canada). Individual frames were recorded at 10 minute intervals and analyzed using ImageJ (http://rsb.info.nih.gov/ij/) and the manual tracking plugin. To improve the contrast of the images and better identify the moving cells, the initial frame was subtracted from all subsequent frames. Tracking was terminated as soon as the cell stopped migrating or reversed its direction. The collected data up until that point was imported into Microsoft Excel where the velocity of the cell was calculated using the distance achieved during the time of forward migration. The velocity and the time to cessation or reversal of movement were analyzed using the total amount of minutes until initial stop. The duration of the movement was calculated based on the time interval between the recorded individual frames. A total of 428 cells were tracked (control cells, 203; DYT1 cells, 225).

2.6. Live cells labeling

Viable fibroblasts were stained with Hoechst 33258 and BODIPY FL C5-ceramide dyes (both from Molecular Probe Inc., Eugene, OR), or PKH67 (Sigma-Aldrich), according to the manufacturer's instructions. Green-fluorescent BODIPY FL C5-ceramide localizes to the Golgi apparatus, blue-fluorescent Hoechst 33258 dye stains the nuclei, and the green fluorescent dye PKH67 stains cell membranes.

2.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 3.0). Results are expressed as mean ± S.E.M. Multiple comparison analysis of variance between the groups was performed by a one-way ANNOV test follow tukey's Multipl Comparison test (MC )/or Bonferoni's Multipl Comparrson test to detrmine if velocity velocity between control and DYT1 fibroblasts were significant during migration. The same was done for migration distance (data not shown), Golgi localization and centrosome orientation. A Spearman correlation was done between the velocity (μm/min) and the time of initial stop (min) between the control and DYT1 fibroblasts.

3. Results

3.1. Migration stopper assay

In initial experiments the migration of DYT1 and control fibroblasts was evaluated using the OrisTM stopper wells. These wells have the advantage that they create a more precisely delineated cell-free zone to evaluate movement of cells over time as compared to the “hand cut” wound zones used in some assays (Araki-Sasaki et al., 1995). However, it is not a measure of vectorial cell movement as cells can migrate in different directions around the margin. The extent of migration into the cell-free zone over 72 hours was assessed by fluorescent staining of cells for an ER marker and quantitation of the fluorescence intensity (Fig. 1A). The random movement of cells around the detection zone/exclusion edge is notable at a higher magnification (Fig. 1B). In the post-migration wells the average fluorescence signal for control fibroblasts was 5,333 (± 351 S.D.) and for patient fibroblasts was 3,700 (± 473 S.D.) (Fig. 1C; p<0.002). This represents an approximately 36% decrease in the extent of cell migration in patient fibroblasts expresssion endgenous torsion ΔE, as compared to control cells, consistent with previous reports using the hand cut wound assay for cells lacking torsinA (Nery et al., 2008).

Fig. 1. Cell migration of control and patient fibroblasts evaluated using fluorescent stain.

Fig. 1

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Cell migration of control (HF77) and DYT1 fibroblasts (2551) was evaluated in the OrisTM stopper assay. A) Fibroblasts were plated at a final concentration of 2.5 x 104 per well in 96-well plates. Twenty-four hours after plating the stoppers were removed - creating a cell-free zone (dotted lines). Cells were fixed and stained by immunocytochemistry for PDI for fluorescence quantification of cells in this zone using a confocal microscope. The images were captured and the data analyzed from pre-migration (t = 0) and post-migration (t = 72 hours). B) High magnification of the “cell-free zone” for control and DYT1 fibroblasts. C) The average fluorescence signal from the detection zones (wound) was calculated as the mean ± S.D. for each condition (n=8 wells/cell type per experiment, from two experiments). In the post-migration wells the average fluorescence signal for control fibroblasts was greater 5333 (± 351 S.D.), as compared to patient fibroblasts 3700 (± 473 S.D.) (p<0.002; n=3 experiments), indicating reduced migration in patient fibroblasts.

3.2. Centrosome orientation assay

At the onset of cell migration the nucleus moves to a position posterior to the centrosome with respect to the leading edge of movement (Pouthas et al., 2008). Centrosome reorientation in cells is rate limiting in cell migration. It has been demonstrated that in fibroblasts polarizing for migration into wounds in culture, an actin-dependent nuclear movement is triggered by serum or the phospholipid serum factor, LPA, and this reorients the centrosome toward the leading edge (Gomes et al., 2005). Using a wound assay, as described (Starr, 2009), confluent monolayers were incubated in serum-free medium for 48 hours, then a hand-cut wound was made in the monolayer, and cells were stimulated with 10 μM LPA (Gomes and Gundersen, 2006). After 3 hours incubation with LPA, we assessed the relative position of the nucleus, centrosome and wound margin by staining for the nucleus, centrosome and microtubules (Fig. 2A). Quantitation was carried out on three control lines and four DYT1 lines with significantly different ranges of 66-76% (average 68%) for correctly oriented nuclei for control cells and 25-34% (average 30%) or DYT1 cells (Fig. 2B; p<0.01 between groups).

Fig. 2. Nuclear movement is disturbed by torsinAΔE.

Fig. 2

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Fibroblasts were treated with or without LPA for 3 hours after wound introduction on monolayer cultures on coverslips, then fixed with 4% PFA and methanol for immunohistochemistry. A) Diagram (top left) indicates oriented vs. non-oriented nucleus (N) with respect to the centrosome (yellow, C) and the wound edge. Staining (bottom left) shows alignment of cells along the wound edge for human control (HF 19) and DYT1 (2551) fibroblasts with centrosome (anti-pericentrin, yellow), nucleus (DAPI, blue) and microtubules (α-tubulin; green). The wound edge is toward the top in both images. Scale bars 10 μm. B) The criteria used for quantification of centrosome orientation were as follows: cells touching the wound edge with the centrosome localized in the sector between the wound and leading edge in front of the nucleus were scored as oriented and centrosomes in other sectors were scored as non-oriented. The % of cells with oriented centrosomes relative to the wound edge and nucleus was significantly different (p<0.01; n=3 experiments) in control and DYT1 fibroblasts.

3.3. Microfluidic chamber assays

In order to obtain more precise measurements of the vectorial movement of cells, microfluidic chambers were prefabricated to contain a well for cell loading and channels radiating out from it, having 10 x 10 μm cross section and 600 μm length to allow single file migration of cells and imaging in real-time (Fig. 3A). Cells were labeled with the PKH67 membrane dye to monitor their extent and position within the chambers over time (Fig. 3B). Time-lapse image analysis was used to determine the forward velocity of four control cell lines and three DYT1 cell lines after they had entered the channels. Movement of individual cells was monitored visually and terminated whenever cells stopped or reversed their direction of movement. DYT1 cells showed an average 58% slower rate of forward movement within channels, as compared to control cells (Fig. 3C). This, and a greater tendency of the DYT1 cells to stop and/or reverse the direction of movement, correlated with the velocity (Fig. 3D) and reduced the total distance traveled by DYT1, as compared to control cells over a fixed period of time.

Fig. 3. Decreased velocity of DYT1 patient fibroblasts in microfluidic chambers.

Fig. 3

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A) Control and DYT1 cells were plated in microfluidic devices positioned inside multiwell plates. Arrays of migration channels allowed the monitoring of single cell migration. B) The fibroblasts were stained with vital green fluorescent membrane dye PKH67 in microchambers. Fibroblast migration was evaluated with high resolution by time-lapse confocal microscopy at 37°C in cell culture chambers. C) The forward velocity of cells was monitored by time-lapse video microscopy. Single-cell tracking of the control human fibroblasts: HF19, n=72 cells; 2131, n=50; HF77, n=30; HF71, n=51 and DYT1 fibroblasts: 2551, n=100; 2306, n=100; HF49, n=25 were obtained over a 24 hour period. The average forward velocity of control cells was 0.24 μm/min, 95% confidence interval (CI)=0.18 to 0.34 μm/min, and DYT1 cells DYT1 0.14 μm/min, 95% CI=0.12 to 0.20 μm/min with a significantly decreased rate of forward movement of DYT1 compared to control cells (p<0.001; n=6 experiments). D) Correlation between the velocity (μm/min) and time to initial stop/reversal of movement (min). The human fibroblasts used were: controls - HF19, 2131, HF77, HF71 and DYT1 fibroblasts - 2551, 2306, HF49. There was a significant positive correlation between the migration velocity of control cell lines (blue), mean=0.25, S.E.M. ± 0.014 versus and DYT1 cells (red), mean=0.14, S.E.M. ± 0.008, and the time to initial stop/reversal of movement (controls, mean=1,072, S.E.M. ± 57.4, DYT1s cells, mean=548.7, S.E.M. ± 43.4), correlation r=0.78, p<0.04.

Microfluidic chambers were also used to evaluate the relative position of cell nuclei and the Golgi apparatus within cells moving inside the channels. Golgi and nuclei were stained with intravital dyes and their relative position was assessed in living migrating control and DYT1 cells (Fig. 4A). Normally the Golgi moves in front of the nucleus under these conditions, but in some cases falls behind the nucleus or appears to surround it. In 75% of control cells (line 2131, n=40) the Golgi was positioned in front of the nucleus with respect to the direction of movement, while in DYT1 cells this dropped significantly to 44% (line 2551, n=57 cells) and 53% (line 2306, n=40 cells) (Fig. 4B; p<0.001).

Fig. 4. Position of Golgi during migration of human control and DYT1 fibroblasts in microfluidic chambers.

Fig. 4

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A) The position of the Golgi complex in human fibroblasts (2131) during the time-lapse microscopy is shown with the directions of movement indicated by arrows. The Golgi complex is stained green (BODIPY-Ceramide, Molecular Probes) and the nucleus, blue (Hoechst 33342, Pierce, Rockford, IL). B) Positions of the Golgi complex (in %) in relation to the nucleus in migrating control (2131) and DYT1 (2551 & 2306) fibroblast lines, indicate whether the Golgi was ahead of or behind the nucleus, or in an undefined position relative to the nucleus. There was a significant difference between the % of cells with the Golgi ahead in control and DYT1 groups (*** p<0.001; n=2 experiments).

4. Discussion

This study takes advantage of the high precision of measuring the speed and persistence of migrating cells through micron sized channels and the visual, real-time live cell monitoring at cellular and subcellular resolution. The mechanical confinement of fibroblasts inside channels of cross-section size comparable to that of the moving cells enables the cells to migrate for long time, at constant speed, unperturbed by interactions with other cells. These migration patterns are distinct from the traditional “random-biased walk” that is usually observous in the traditional methods used for measuring cell migration on flat surfaces, like the conventional stopper and wound healing methods, or the large majority of microfluidic devices for cell migration when cell migration also takes place on flat surfaces. Unlike the migration on flat surfaces, when the characteristic speed and directionality of migrating cells changes often, are perturbed by cell-cell interactions, and can only be estimated with limited precision by statistical methods, when inside channels, cells move at uniform speed and change direction far less frequently, enabling direct and precise measurements of migration parameters at single cell resolution. Taking advantage of the increased precision of the measurements, we observed and quantified the distinct motility behavior of DYT1 and wild-type fibroblasts. In addition, microfluidic devices permit single cell analysis as they are compatible with modern microscopic methods. The vectorial cell movement through channels helped significantly the study of movement of organelles inside the moving cells by providing a predictable reference frame for the position of the nucleus and leading edge at any time.

In addition, our findings confirm previous studies (Nery et al., 2008) indicating a reduced rate of migration of fibroblasts from DYT1 dystonia patients as compared to controls, due at least in part to a decreased nuclear polarization with respect to the centrosome and the leading edge of migration. Using microfluidic chambers we were able to show that the velocity and distance covered is also reduced in DYT1, as compared to control cells. In addition, DYT1 cells have a greater tendency to pause in migration and reverse direction. This also may explain an additional abnormal feature in which the relative positions of the Golgi and nucleus is more variable in DYT1, as compared to control cells in the process of migrating.

The longitudinal asymmetry of cellular structures relative to the nucleus is a common characteristic of various polarized cells. This asymmetry appears to be important during cell migration. For example, in epithelial cells the microtubule organization complex is often localized between the nucleus and leading edge (Yvon et al., 2002) and the ER and Golgi apparatus also occupy similar anterior positions (Miller et al., 2009; Pouthas et al., 2008). In moving lymphocytes, mitochondria accumulate behind the nucleus, toward the uropod, with consequences on the increased persistence of migration in a specific direction (Campello et al., 2006), and share asymmetrical localization with the microtubule organizing center (del Pozo et al., 1998). The effect of asymmetric organelle distribution inside moving cells can be precisely quantified when cell migration is confined in small channels, e.g. the anterior-localized mitochondria could account for an order of magnitude difference in invasive abilities between cells from otherwise homogenous cell populations (Desai et al., 2013). For moving fibroblasts, confinement along channels accentuates the correlations between intracellular organization and migration parameters, circumventing the limitations of studies on flat surfaces when cells change directions often and can extend multiple pseudopods at the same time (Uetrecht and Bear, 2009).

These studies are consistent with the findings that torsinA, located in the lumen of the contiguous NE and ER, has a role in morphology and orientation of the nucleus and that mutant torsinA interferes with this orientation (Naismith et al., 2004; Goodchild and Dauer, 2004; Nery et al., 2008). This function is due, at least in part, to associations between torsinA and the nesprin proteins which span the outer nuclear membrane and interact with different cytoskeletal elements in the cytoplasm, as well as SUN proteins that span the inner nuclear membrane and interact with nesprins in the lumen of the NE (Nery et al., 2008; Atai et al., 2012; Jungwirth et al., 2011; Vander Heyden et al., 2009). Since torsinA is expressed in most cells of the body the effects of mutant torsinA can be assessed in fibroblasts which can be obtained from patients, and normal torsinA can be assessed in fibroblasts from healthy, unaffected controls. Our current findings support both an initial delay in onset of migration in the culture wound paradigm as well as reduced forward velocity in primary DYT1 as compared to control cells, both of which may be explained by compromised relationship between the nuclear membrane and the cytoskeleton. Importantly, this is consistent with delayed migration of neurons during embryonic brain development in DYT1 torsinA knock-out mice both in culture and in vivo (McCarthy et al., 2012). Other studies have shown an altered morphology of neurons in DYT1 heterozygous knock-in mouse brains (Song et al., 2013; Zhang et al., 2011) and of the NE in neurons in DYT1 homozygous knock-out or knock-in brains (Goodchild et al., 2005). In parallel, dtorsin-null Drosophila has abnormal morphology of dopaminergic neurons and patterning of their axons in adult brains (Wakabayashi-Ito et al., 2011). Thus, a contribution to the pathophysiology of DYT1 dystonia may lie in subtle alterations in brain microcircuitry resulting from abnormalities in the association of the NE and ER with the cytoskeleton leading to reduced migration and extension of neuronal process in subsets of neurons during development.

5. Conclusion

These studies show that the microfluidic chambers designed to evaluate single cell migration in real-time provide a highly quantitative means for analysis of vectorial movement of cells, including positioning of organelles within migrating cells. We also confirm that cells expressing endogenous levels of both mutant and wild-type torsinA (from DYT1 patients) have a reduced rate of migration and misorientation of the nucleus during migration, as compared to control cells that express only wild-type torsinA.

Highlights.

  • Microfluidic chambers provide a highly quantitative means for analysis of vectorial movement of cells, including positioning of organelles within migrating cells.

  • Reduced rate of migration was observed in fibroblasts from DYT1 dystonia patients as compared to controls.

  • Abnormal feature in the relative positions of the Golgi and nucleus is more variable in DYT1, as compared to control cells in the process of migrating.

Acknowledgements

The authors thank Ms. Suzanne McDavitt for skilled editorial assistance. This work was supported by NIH/NINDS Grants NS037409 (XOB) and GM092804 (DI); Parikinson's n Movement Disorder Foundation and Harvard Medical School The Eleanor and Miles Shore 50th Anniversary Fellowship Program for Scholars in Medicine (FCN); AMC Medical/Graduate School Ph.D. Scholarships, University of Amsterdam, The Netherlands (NAA). We also thank you the Confocal Core Facility at Neuroscience Center MGH P30 NS045776 NIH/NINDS.

Abbreviations

DMEM

Dulbe co's Modi Eagle's Medium

ER

endoplasmic reticulum

LPA

lysophosphatidic acid

MCT

Multiple Comparison test

NE

nuclear envelope

PBS

phosphate buffered saline

PDI

protein disulfide isomerase

PDMS

polydimethyl siloxane

PFA

paraformaldehyde

Footnotes

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Conflict of interest

Authors have no conflict of interest.

References

  1. Albanese A, Lalli S. Update on dystonia. Curr Opin Neurol. 2012;25:483–90. doi: 10.1097/WCO.0b013e3283550c22. [DOI] [PubMed] [Google Scholar]
  2. Albanese A, Bhatia K, Bressman SB, Delong MR, Fahn S, Fung VS, Hallett M, Jankovic J, Jinnah HA, Klein C, Lang AE, Mink JW, Teller JK. Phenomenology and classification of dystonia: a consensus update. Mov Disord. 2013;28:863–73. doi: 10.1002/mds.25475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ambravaneswaran V, Wong IY, Aranyosi AJ, Toner M, Irimia D. Directional decisions during neutrophil chemotaxis inside bifurcating channels. Integr Biol (Camb) 2010;2:639–47. doi: 10.1039/c0ib00011f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H. An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci. 1995;36:614–21. [PubMed] [Google Scholar]
  5. Atai NA, Ryan SD, Kothary R, Breakefield XO, Nery FC. Untethering the nuclear envelope and cytoskeleton: biologically distinct dystonias arising from a common cellular dysfunction. Int'l J Cell Biology. 2012;2012:634214. doi: 10.1155/2012/634214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Augood SJ, Penney JB, Jr., Friberg IK, Breakefield XO, Young AB, Ozelius LJ, Standaert DG. Expression of the early-onset torsion dystonia gene (DYT1) in human brain. Ann Neurol. 1998;43:669–73. doi: 10.1002/ana.410430518. [DOI] [PubMed] [Google Scholar]
  7. Augood SJ, Martin DM, Ozelius LJ, Breakefield XO, Penney JB, Jr., Standaert DG. Distribution of the mRNAs encoding torsinA and torsinB in the normal adult human brain. Ann Neurol. 1999;46:761–9. [PubMed] [Google Scholar]
  8. Augood SJ, Hollingsworth ZR, Standaert DG, Emson PC, Penney JB. Localization of dopaminergic markers in the human subthalamic nucleus. J Comp Neurol. 2000;421:247–55. [PubMed] [Google Scholar]
  9. Bragg DC, Armata IA, Nery FC, Breakefield XO, Sharma N. Molecular pathways in dystonia. Neurobiol Dis. 2011;42:136–47. doi: 10.1016/j.nbd.2010.11.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Breakefield XO, Braverman M, Riker DK, Giller ELJ. Catechol-O-methyltransferase activity in cultured human skin fibroblasts from controls and patients with dystonia musculorum deformans. J Neurosci Res. 1981;6:349–60. doi: 10.1002/jnr.490060310. [DOI] [PubMed] [Google Scholar]
  11. Bressman SB, Raymond D, Wendt K, Saunders-Pullman R, De Leon D, Fahn S, Ozelius L, Risch N. Diagnostic criteria for dystonia in DYT1 families. Neurology. 2002;59:1780–2. doi: 10.1212/01.wnl.0000035630.12515.e0. [DOI] [PubMed] [Google Scholar]
  12. Burbulla LF, Krüger R. The use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of Parkinson's disease. J Vis Exp. 2012;3:4228. doi: 10.3791/4228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Butler KL, Ambravaneswaran V, Agrawal N, Bilodeau M, Toner M, Tompkins RG, Fagan S, Irimia D. Burn injury reduces neutrophil directional migration speed in microfluidic devices. PLoS One. 2010;5:e11921. doi: 10.1371/journal.pone.0011921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Campello S, Lacalle RA, Bettella M, Mañes S, Scorrano L, Viola A. Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J Exp Med. 2006;203:2879–86. doi: 10.1084/jem.20061877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cao S, Hewett JW, Yokoi F, Lu J, Buckely AC, Burdette AJ, Chen P, Nery FC, Li Y, Breakefield XO, Caldwell GA, Caldwell KA. Chemical enhancement of torsinA function in cell and animal models of torsion dystonia. Dis Models Mech. 2010;3:386–96. doi: 10.1242/dmm.003715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chen P, Burdette AJ, Porter CJ, Ricketts JC, Fox SA, Hewett JW, Nery FC, Berkowitz LA, Breakefield XO, Caldwell KA, Caldwell GA. The early-onset torsion dystonia associated protein, torsinA, is a homeostatic regulator of endoplasmic reticulum stress response. Hum Mol Genet. 2010;19:3502–15. doi: 10.1093/hmg/ddq266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cho H, Hashimoto T, Wong E, Hori Y, Wood LB, Zhao L, Haigis KM, Hyman BT, Irimia D. Microfluidic chemotaxis platform for differentiating the roles of soluble and bound amyloid-β on microglial accumulation. Sci Rep. 2013;3:1823. doi: 10.1038/srep01823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Connolly GP. Fibroblast models of neurological disorders: fluorescence measurement studies. Trends Pharmacol Sci. 1998;19:171–7. doi: 10.1016/s0165-6147(98)01202-4. [DOI] [PubMed] [Google Scholar]
  19. del Pozo MA, Nieto M, Serrador JM, Sancho D, Vicente-Manzanares M, Martínez C, Sánchez-Madrid F. The two poles of the lymphocyte: specialized cell compartments for migration and recruitment. Cell Adhes Commun. 1998;6:125–33. doi: 10.3109/15419069809004468. [DOI] [PubMed] [Google Scholar]
  20. Desai SP, Bhatia SN, Toner M, Irimia D. Mitochondrial localization and the persistent migration of epithelial cancer cells. Biophys J. 2013;104:2077–88. doi: 10.1016/j.bpj.2013.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fahn S. The varied clinical expressions of dystonia. Neurol Clin. 1984;2:541–54. [PubMed] [Google Scholar]
  22. Falkenburger BH, Schulz JB. Limitations of cellular models in Parkinson's disease research. J Neural Transm Suppl. 2006;70:261–8. doi: 10.1007/978-3-211-45295-0_40. [DOI] [PubMed] [Google Scholar]
  23. Ferraiuolo L, Meyer K, Kaspar B. Current Advances in Amyotrophic Lateral Sclerosis. Intech Open Science; 2013. The use of human samples to study familial and sporadic amyotrophic lateral schlerosis: New frontiers and challenges. pp. 159–182. Chapter 6. [Google Scholar]
  24. Fung VS, Jinnah HA, Bhatia K, Vidailhet M. Assessment of patients with isolated or combined dystonia: an update on dystonia syndromes. Mov Disord. 2013;28:889–98. doi: 10.1002/mds.25549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gibbs JR, Singleton A. Application of genome-wide single nucleotide polymorphism typing: simple association and beyond. PLoS Genet. 2006;2:e150. doi: 10.1371/journal.pgen.0020150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gomes ER, Jani S, Gundersen GG. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell. 2005;121:451–63. doi: 10.1016/j.cell.2005.02.022. [DOI] [PubMed] [Google Scholar]
  27. Gomes ER, Gundersen GG. Real-time centrosome reorientation during fibroblast migration. Methods Enzymol. 2006;406:579–92. doi: 10.1016/S0076-6879(06)06045-9. [DOI] [PubMed] [Google Scholar]
  28. Goodchild RE, Dauer WT. Mislocalization to the nuclear envelope: an effect of the dystonia-causing torsinA mutation. Proc Natl Acad Sci U S A. 2004;101:847–52. doi: 10.1073/pnas.0304375101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Goodchild RE, Kim CE, Dauer WT. Loss of the dystonia-associated protein torsinA selectively disrupts the neuronal nuclear envelope. Neuron. 2005;48:923–32. doi: 10.1016/j.neuron.2005.11.010. [DOI] [PubMed] [Google Scholar]
  30. Hewett JW, Tannous B, Niland BP, Nery FC, Breakefield XO. Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells. Proc Natl Acad Sci U S A. 2007;104:7271–6. doi: 10.1073/pnas.0701185104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Irimia D, Toner M. Spontaneous migration of cancer cells under conditions of mechanical confinement. Integr Biol (Camb) 2009;1:506–12. doi: 10.1039/b908595e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jokhi V, Ashley J, Nunnari J, Noma A, Ito N, Wakabayashi-Ito N, Morre MJ, Budnik V. Torsin mediates primary envelopment of large ribonucleoprotein granules at the nuclear envelope. Cell Rep. 2013;3:988–95. doi: 10.1016/j.celrep.2013.03.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jungwirth MT, Kumar D, Jeong DY, Goodchild RE. The nuclear envelope localization of DYT1 dystonia torsinA-ΔE requiries the SUN1 LINC complex component. BMC Cell Biol. 2011;12:24. doi: 10.1186/1471-2121-12-24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Koch P, Breuer P, Peitz M, Jungverdorben J, Kesavan J, Poppe D, Doerr J, Ladewig J, Mertens J, Tüting T, Hoffmann P, Klockgether T, Evert BO, Wüllner U, Brüstle O. Excitation-induced ataxin-3 aggregation in neurons from patients with Machado-Joseph disease. Nature. 2011;480:543–6. doi: 10.1038/nature10671. [DOI] [PubMed] [Google Scholar]
  35. Kurosaka S, Kashina A. Cell biology of embryonic migration. Birth Defects Res C Embryo Today. 2008;84:102–22. doi: 10.1002/bdrc.20125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lohmann K, Klein C. Genetics of dystonia: what's known? What's new? What's next? Mov Disord. 2013;28:899–905. doi: 10.1002/mds.25536. [DOI] [PubMed] [Google Scholar]
  37. Maric M, Shao J, Ryan RJ, Wong CS, Gonzalez-Alegre P, Roller RJ. A functional role for TorsinA in herpes simplex virus 1 nuclear egress. J Virol. 2011;85:9667–79. doi: 10.1128/JVI.05314-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. McCarthy DM, Gioioso V, Zhang X, Sharma N, Bhide PG. Neurogenesis and neuronal migration in the forebrain of the torsinA knockout mouse embryo. Dev Neurosci. 2012;34:366–78. doi: 10.1159/000342260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Miller PM, Folkmann AW, Maia AR, Efimova N, Efimov A, Kaverina I. Golgi-derived CLASP-dependent microtubules control Golgi organization and polarized trafficking in motile cells. Nat Cell Biol. 2009;11:1069–80. doi: 10.1038/ncb1920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Naismith TV, Heuser JE, Breakefield XO, Hanson PI. TorsinA in the nuclear envelope. Proc Natl Acad Sci U S A. 2004;101:7612–7. doi: 10.1073/pnas.0308760101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nery FC, Zeng J, Niland BP, Hewett J, Farley J, Irimia D, Li Y, Wiche G, Sonnenberg A, Breakefield XO. TorsinA binds the KASH-domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton. J Cell Sci. 2008;121:3476–86. doi: 10.1242/jcs.029454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nery FC, Armata IA, Farley JE, Cho JA, Yaqub U, Chen P, da Hora CC, Wang Q, Tagaya M, Klein C, Tannous B, Caldwell KA, Caldwell GA, Lencer WI, Ye Y, Breakefield XO. TorsinA participates in endoplasmic reticulum-associated degradation. Nat Commun. 2011;2:393. doi: 10.1038/ncomms1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Palazzo AF, Joseph HL, Chen YJ, Dujardin DL, Alberts AS, Pfister KK, Vallee RB, Gundersen GG. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr Biol. 2001;11:1536–41. doi: 10.1016/s0960-9822(01)00475-4. [DOI] [PubMed] [Google Scholar]
  44. Pouthas F, Girard P, Lecaudey V, Ly TB, Gilmour D, Boulin C, Pepperkok R, Reynaud EG. In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum. J Cell Sci. 2008;121:2406–14. doi: 10.1242/jcs.026849. [DOI] [PubMed] [Google Scholar]
  45. Qiang L, Fujita R, Yamashita T, Angulo S, Rhinn H, Rhee D, Doege C, Chau L, Aubry L, Vanti WB, Moreno H, Abeliovich A. Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons. Cell. 2011;146:359–71. doi: 10.1016/j.cell.2011.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  46. Quartarone A, Pisani A. Abnormal plasticity in dystonia: Disruption of synaptic homeostasis. Neurobiol Dis. 2011;42:162–70. doi: 10.1016/j.nbd.2010.12.011. [DOI] [PubMed] [Google Scholar]
  47. Rostasy K, Augood SJ, Hewett JW, Leung JC, Sasaki H, Ozelius LJ, Ramesh V, Standaert DG, Breakefield XO, Hedreen JC. TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion. Neurobiol Dis. 2003;12:11–24. doi: 10.1016/s0969-9961(02)00010-4. [DOI] [PubMed] [Google Scholar]
  48. Scherber C, Aranyosi AJ, Kulemann B, Thayer SP, Toner M, Iliopoulos O, Irimia D. Epithelial cell guidance by self-generated EGF gradients. Integr Biol (Camb) 2012;4:259–69. doi: 10.1039/c2ib00106c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Song CH, Bernhard D, Hess EJ, Jinnah HA. Subtle microstructural changes of the cerebellum in a knock-in mouse model of DYT1 dystonia. Neurobiol Dis. 2013;62C:372–80. doi: 10.1016/j.nbd.2013.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Starr DA. A nuclear-envelope bridge positions nuclei and moves chromosomes. J Cell Sci. 2009;122:577–86. doi: 10.1242/jcs.037622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Uetrecht AC, Bear JE. Golgi polarity does not correlate with speed or persistence of freely migrating fibroblasts. Eur J Cell Biol. 2009;88:711–7. doi: 10.1016/j.ejcb.2009.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vander Heyden AB, Naismith TV, Snapp EL, Hodzic D, Hanson PI. LULL1 retargets TorsinA to the nuclear envelope revealing an activity that is impaired by the DYT1 dystonia mutation. Mol Biol Cell. 2009;20:2661–72. doi: 10.1091/mbc.E09-01-0094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vasudevan A, Breakefield XO, Bhide P. Developmental patterns of torsinA and torsinB expression. Brain Res. 2006;1073-1074:139–45. doi: 10.1016/j.brainres.2005.12.087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wakabayashi-Ito N, Doherty OM, Moriyama H, Gusella JF, O'Donnell JM, Ito N. dtorsin, the Drosophila ortholog of the early-onset dystonia TOR1A (DYT1), plays a novel role in the dopamine metabolism. PLoS One. 2011;6:e26183. doi: 10.1371/journal.pone.0026183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart S, Slukvin SI, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917–20. doi: 10.1126/science.1151526. [DOI] [PubMed] [Google Scholar]
  56. Yvon AM, Walker JW, Danowski B, Fagerstrom C, Khodjakov A, Wadsworth P. Centrosome reorientation in wound-edge cells is cell type specific. Mol Biol Cell. 2002;13:1871–80. doi: 10.1091/mbc.01-11-0539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang L, Yokoi F, Jin YH, DeAndrade MP, Hashimoto K, Standaert DG, Li Y. Altered dendritic morphology of Purk cells in Dyt1 ΔGAG knock-in and purkinje cell-specific Dyt1 conditional knockout mice. PLoS One. 2011;6:e18357. doi: 10.1371/journal.pone.0018357. [DOI] [PMC free article] [PubMed] [Google Scholar]

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