Role of cytoskeleton in controlling the disorder strength of cellular nanoscale architecture

Abstract

Cytoskeleton is ubiquitous throughout the cell and is involved in important cellular processes such as cellular transport, signal transduction, gene transcription, cell-division, etc. Partial wave spectroscopic microscopy is a novel optical technique that measures the statistical properties of cell nanoscale organization in terms of the disorder strength. It has been found previously that the increase in the disorder strength of cell nanoarchitecture is one of the earliest events in carcinogenesis. In this study, we investigate the cellular components responsible for the differential disorder strength between two morphologically (and hence microscopically) similar but genetically altered human colon cancer cell lines, HT29 cells and Csk shRNA-transfected HT29 cells that exhibit different degrees of neoplastic aggressiveness. To understand the role of cytoskeleton in nanoarchitectural alterations, we performed selective drug treatment on the specific cytoskeletal components of these cell types and studied the effects of cytoskeletal organization on disorder strength differences. We report that altering the cell nanoarchitecture by disrupting cytoskeletal organization leads to the attenuation of the disorder strength differences between microscopically indistinguishable HT29 and CSK constructs. We therefore demonstrate that cytoskeleton plays a role in the control of cellular nanoscale disorder.

Figure 1

Schematic of the second generation PWS system. A1, A2, apertures [D(A1) = 2 mm, D(A2) = 4 mm]; BS, beam splitter; C, condenser; L1, L2, lenses (f = 150 mm); F, flipper mirror; LCTF, liquid crystal tunable filter; M, mirror; OBJ, objective lens; SS, sample stage; TL, tube lens (f = 450 mm).

Figure 2

Representative confocal images of HT29 and CSK cells. (a and c) Three-dimensional top view of the HT29 cells and CSK constructs respectively. These cells appear microscopically and morphologically similar. The red region represents nucleus being stained with TOPRO3 (nuclear chromatin stain). The green region represents cytoplasm stained with anti-β-tubulin. (b and d) Slice cut through the cell in the x-z plane. There appears to be negligible cytoplasm (∼10–15%) on top and bottom of the nucleus. (e and f) Results of PWS analysis carried out independently on nuclear and cytoplasmic region of both the cell constructs respectively. There appears statistically highly significant difference (p < 0.00001) in the average disorder strength, calculated from the nuclear region of HT29 cells and CSK constructs. Also, there is a significant (p < 0.05) difference, ΔL_d observed in cytoplasmic region of these cell constructs.

Figure 3

(a) Disorder strength (L_d) quantification for the nucleus of cytochalasin D-treated HT29 and CSK constructs show almost equal average disorder strength values. The ΔL_d is statistically not significant (p = 0.54). This can be attributed to the effect of cytochalasin D on nuclear actin. (b) The ΔL_d for cytoplasmic region of HT29 and CSK constructs is also minimal and is statistically nonsignificant (p = 0.66) that may be attributed to the rupture of filamentous actin in the cytoplasm.

Figure 4

(a) Treatment with colchicine renders statistically significant (p = 0.002) ΔL_d for the nuclear region of HT29 cells and CSK constructs as there are no microtubules present in the nucleus of these undividing cells. (b) The ΔL_d reduces significantly (p = 0.87) in the cytoplasm of the colchicine-treated HT29 and CSK constructs that could be attributed to the rupture of microtubules present only in the cytoplasm.