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The Role of the Cytoskeleton in Structurally Maintaining Vacuoles of Notochordal Cells
Lauren Resutek1, Adam H. Hsieh1, 2
University of Maryland, College Park, MD, 2University of Maryland, Baltimore, MD
1
DISCLOSURES: The authors have nothing to disclose.
INTRODUCTION: Intervertebral disc (IVD) degeneration is a leading cause of low back pain (LBP), a common and often debilitating condition associated
with an estimated annual cost of over $100 billion.1,2 As humans age, the IVD becomes more susceptible to degeneration due to compositional and
subsequent functional changes. For instance, the nucleus pulposus (NP) tissue of the IVD experiences decreases in water and proteoglycan content impairing
its ability to generate and sustain hydrostatic pressure – a prominent stress experienced in vivo. Simultaneously, cells of the NP tend to transition from
immature, notochordal cells (NCs) to mature, chondrocyte-like NP cells. Because NP cells are responsible for the synthesis of a functional extracellular
matrix, this cellular transition is believed to be involved in the progression of disc degeneration. Morphologically, NCs can be differentiated from mature NP
cells by their larger size and abundance of cytosolic vacuoles. Another defining feature of NCs is their co-expression of vimentin and cytokeratin
intermediate filaments, where mature NP cells typically only express vimentin. Using a human chordoma cell line as a model for NCs, this investigation
seeks to elucidate the involvement of the cytoskeleton in maintaining vacuoles characteristic of NCs. Because the loss of vacuoles in mature NP cells
coincides with changes in intermediate filament expression, we hypothesize intermediate filaments are involved in structurally supporting NC vacuoles.
METHODS: Cell Culture: MUG-Chor1 (ATCC CRL-3219) chordoma cells were cultured in chordoma medium (Iscove's Modified Dulbecco's Medium:
Roswell Park Memorial Institute 1640 Medium (4:1) supplemented with 10% FBS and 1% Penicillin/Streptomycin) on type I collagen coated cover glass.
Immunofluorescence: Cells were stained with calcein AM, a live stain that does not permeate into the vacuole lumen, fixed, and labeled with either anticytokeratin-8 [M20] (1:100 Abcam), anti-vimentin [SP20] (1:200 Thermo Scientific), or anti--tubulin polyclonal (1:200 Abcam) antibodies or Alexa
Fluor594 Phalloidin (1:40 Invitrogen, Inc.), tagging F-actin. Cells were imaged using confocal microscopy with a Nipkow (spinning) disk-equipped
Olympus IX81 microscope. Cytoskeletal Disruption: Cytochalasin-D (1M), nocodazole (5M), and acrylamide (40mM) were diluted in chordoma medium
and separately incubated with cells to disrupt F-actin, microtubules, and intermediate filaments, respectively. Cytochalasin-D and nocodazole were incubated
with cells for 1h and acrylamide 3.5h. Disruption of targeted cytoskeletal elements was verified using immunofluorescence and confocal microscopy.
Vacuole Analysis: To verify cell viability following cytoskeletal disruption, cells were stained with calcein AM (green) and ethidium homodimer (red). For
quantitative analysis, vacuoles were manually counted using phase-contrast images of the same cells before and after cytoskeletal disruption. Control cells
were imaged before and 3.5h after the addition of untreated medium. For each group, the non-parametric Wilcoxon test was used to compare the number of
vacuoles before and after treatment. Additionally, Kruskal-Wallis followed by Mann-Whitney U tests were used to compare the percent vacuole loss
between groups.
RESULTS: Comparing calcein and immunofluorescence images revealed a relationship between cytokeratin organization and vacuole location, where
cytokeratin intermediate filaments formed enclosures around vacuoles (Fig. 1A). This relationship was not observed for vimentin intermediate filaments
(Fig. 1B), F-actin (Fig. 1C), or microtubules (Fig. 1D). Disruption of the intermediate filament network with acrylamide resulted in a significant (p<0.0005)
decrease in vacuoles (Fig. 2B, C) and remaining vacuoles appeared collapsed compared to their typical circular morphology (Fig. 2A). While less visually
apparent, disruption of F-actin with cytochalasin-D and microtubules with nocodazole also resulted in a significant (p<0.0005) decrease in vacuoles (Fig.
2B). However, in contrast to the statistical significance observed for the percent vacuole loss of acrylamide-treated cells, no statistical significance was found
comparing the percent vacuole loss of cytochalasin-D-treated, nocodazole-treated, and control cells (Fig. 2C).
DISCUSSION: This study examines the potential role of the cytoskeleton in structurally supporting cytosolic vacuoles characteristic of NCs but not mature
NP cells. The significant decrease in the number of vacuoles following disruption of F-actin, microtubules, and intermediate filaments suggests the
cytoskeleton does in fact play a role in structurally maintaining vacuoles. In support of our hypothesis, the significant percent vacuole loss observed
following the disruption of intermediate filaments, but not F-actin or microtubules, suggests intermediate filaments are primarily involved in the structural
maintenance of vacuoles. Because NCs co-express vimentin and cytokeratin, this suggested role could be attributed to vimentin and/or cytokeratin
intermediate filaments. Based on the observed organization of cytokeratin around vacuoles, we believe cytokeratin, rather than vimentin, intermediate
filaments are required for maintaining vacuoles and the ablation of cytokeratin in mature NP cells is involved in the disappearance of cytosolic vacuoles.
Future studies will seek to elucidate the individual roles of vimentin and cytokeratin intermediate filaments in maintaining NC vacuoles using siRNA
mediated knockdown of vimentin and cytokeratin.
SIGNIFICANCE: Understanding elements that function to stabilize NC vacuoles will give insight into mechanisms of vacuole disappearance and the NP
cellular transition associated with aging and disc degeneration.
REFERENCES: [1] J. N. Katz. J. Bone Joint Surg. Am. 2006. 88: 21-24; [2] D. Hoy, et al. Best Pract Res Clin Rheumatol. 2010. 24(6): 769-781
Fig 1. Chordoma cells stained with calceinAM (green) to observe vacuoles and labeled
for cytoskeletal proteins (red). Cytokeratin8 filaments (A) formed an organized network
around vacuoles. This organization was not
observed for vimentin (B), F-actin (C), or βTubulin (D). Scale bars: 10µm
Fig 2. A. Chordoma cells imaged before and after treatment
with cytoskeleton-disrupting agents. Scale bars: 16µm. B.
The average number of vacuoles before and after treatment
with acrylamide (n=37), cytochalasin-D (n=44), nocodazole
(n=57), or control medium (n=37) reported as avg. ± SEM.
*p<0.001, relative to before acrylamide; +p<0.001, relative
to before cytochalasin-D; #p<0.001, relative to before
nocodazole. C. The average % vacuole loss following
treatment with acrylamide (n=37), cytochalasin-D (n=44),
nocodazole (n=57), or control medium (n=37) reported as
avg. ± SEM. *p<0.001, relative to control, cytochalasin-D
and nocodazole.
ORS 2017 Annual Meeting Poster No.1819