Tao T. Wu

Conserved Motif of CDK5RAP2 Mediates Its Localization to Centrosomes and the Golgi Complex

As the primary microtubule-organizing centers, centrosomes require γ-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a γ-tubulin complex-binding protein and functions in γ-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATP- and centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca2+-independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.

Unified Mie and fractal scattering by cells and experimental study on application in optical characterization of cellular and subcellular structures

A unified Mie and fractal model for light scattering by biological cells is presented. This model is shown to provide an excellent global agreement with the angular dependent elastic light scattering spectroscopy of cells over the whole visible range (400 to 700 nm) and at all scattering angles (1.1 to 165 deg) investigated. Mie scattering from the bare cell and the nucleus is found to dominate light scattering in the forward directions, whereas the random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, is found to dominate light scattering at other angles. Angularly dependent elastic light scattering spectroscopy aided by the unified Mie and fractal model is demonstrated to be an effective noninvasive approach to characterize biological cells and their internal structures. The acetowhitening effect induced by applying acetic acid on epithelial cells is investigated as an example. The changes in morphology and refractive index of epithelial cells, nuclei, and subcellular structures after the application of acetic acid are successfully probed and quantified using the proposed approach. The unified Mie and fractal model may serve as the foundation for optical detection of precancerous and cancerous changes in biological cells and tissues based on light scattering techniques.

Unified Mie and fractal scattering by biological cells and subcellular structures

Angle-resolved light scattering spectroscopy of biological cells is investigated in the visible wavelength range. A unified Mie and fractal model is shown to provide an accurate global agreement with light scattering spectra from 1.1 degrees to 165 degrees scattering angles. It is found that light scattering in forward directions (<8 degrees ) is dominated by Mie scattering by the bare cell and nucleus, whereas light scattering at large angles (>20 degrees ) is determined by fractal scattering by subcellular structures. The findings are consistent with the results of experimental investigation of the contributions of different cellular components to light scattering by cells.

Optical imaging for medical diagnosis based on active stereo vision and motion tracking

This study aims to develop a novel imaging technique to improve the accuracy of the colposcolpic diagnosis of cervical cancer. An optical imaging system based on active stereo vision is built to measure the 3-D surface topology of cervix and track the motion of patient. The information of motion tracking are used to register the time-sequenced images of cervix recorded over the period of examination. The imaging system is evaluated by tracking the movements of cervix models. The results show that the error of 2-D image registration is 0.8 pixels, equivalent to the motion tracking error of 0.05 mm in the field-of-view. The imaging technique holds the promise to enable quantitative mapping of the acetowhitening kinetics over cervical surface for more accurate diagnosis of cervical cancer.

Assessment of the relative contribution of cellular components to the acetowhitening effect in cell cultures and suspensions using elastic light-scattering spectroscopy

We aim to investigate the mechanism of acetowhitening upon which the colposcopic diagnosis of cervical cancer is based. The changes in light scattering induced by acetic acid in intact cervical cancer cells and cellular components were studied using elastic light-scattering spectroscopy. After adding acetic acid to intact cancer cell culture samples (cell suspensions and attached monolayer cell cultures), a slight decrease in small-angle forward scattering was observed, while the large-angle scattering increased by a factor of 5-9, indicating that acetowhitening signals are mainly contributed from small-sized intracellular scattering structures. The cellular components of different sizes and masses were isolated to investigate their individual contribution to the changes of light scattering induced by acetic acid. The study provided the evidence that the cellular components of diameter smaller than 0.2 microm in the cytoplasm are the major contributors to the acetowhitening effect in whole cells, while the light scattering from the mitochondria are not sensitive to the acetic acid.

Study of dynamic process of acetic acid induced-whitening in epithelial tissues at cellular level

Acetic acid, inducing transient whitening (acetowhitening) when applied to epithelial tissues, is a commonly used contrast agent for detecting early cervical cancer. The goals of this research are to investigate the temporal characteristics of acetowhitening process in cervical epithelial tissue at cellular level and develop a clear understanding of the diagnostic information carried in the acetowhitening signal. A system measuring time-resolved reflectance was built to study the rising and decay processes of acetowhitening signal from the monolayered cell cultures of normal and cancerous cervical squamous cells. It is found that the dynamic processes of acetowhitening in normal and cancerous cells are significantly different. The results of this study provide insight valuable to further understand the acetowhitening process in epithelial cells and to encourage the development of an objective procedure to detect the early cervical cancers based on quantitative monitoring of the dynamic process of acetowhitening.

Preliminary study of detecting neoplastic growths in vivo with real time calibrated autofluorescence imaging.

The goal of this study was to evaluate the capabilities of a calibrated autofluorescence imaging method for detecting neoplastic lesions. An imaging system that records autofluorescence images calibrated by the cross-polarized reflection images from excitation was instrumented for the evaluation. Cervical tissue was selected as the living tissue model. Sixteen human subjects were examined in vivo with the imaging system before routine examination procedures. It was found that calibrated autofluorescence signals from neoplastic lesions were generally lower than signals from normal cervical tissue. Neoplastic lesions can be differentiated from surrounding normal tissue based on the contrast in the calibrated autofluorescence. The effects of the optical properties of tissue on the calibrated fluorescence imaging were investigated.

Clinical study of quantitative diagnosis of early cervical cancer based on the classification of acetowhitening kinetics

A quantitative colposcopic imaging system for the diagnosis of early cervical cancer is evaluated in a clinical study. This imaging technology based on 3-D active stereo vision and motion tracking extracts diagnostic information from the kinetics of acetowhitening process measured from the cervix of human subjects in vivo. Acetowhitening kinetics measured from 137 cervical sites of 57 subjects are analyzed and classified using multivariate statistical algorithms. Cross-validation methods are used to evaluate the performance of the diagnostic algorithms. The results show that an algorithm for screening precancer produced 95% sensitivity (SE) and 96% specificity (SP) for discriminating normal and human papillomavirus (HPV)-infected tissues from cervical intraepithelial neoplasia (CIN) lesions. For a diagnostic algorithm, 91% SE and 90% SP are achieved for discriminating normal tissue, HPV infected tissue, and low-grade CIN lesions from high-grade CIN lesions. The results demonstrate that the quantitative colposcopic imaging system could provide objective screening and diagnostic information for early detection of cervical cancer.

Optical imaging of cervical precancerous lesions based on active stereo vision and motion tracking

A 3-D optical imaging system based on active stereo vision and motion tracking is built to track the motion of patient and to register the time-sequenced images of cervix recorded during colposcopy. The imaging system is evaluated by examining human subjects in vivo before routine colposcopy examination procedures. The system tracks the motion of patient accurately. The temporal kinetics of the acetowhitening process in the area of interest can be quantitatively measured. The results demonstrate that the kinetics of acetowhitening may be potentially used for accurately differentiating the precancerous lesions from the normal and benign lesions, and grading the precancerous lesions.

Elastic light scattering by cells: from Mie scattering to fractal scattering

A unified theory for light scattering by biological cells is presented. It is shown that Mie scattering from the bare cell and the nucleus dominates cell light scattering in the forward directions. The random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, dominates light scattering by cells in other angles. The theory is validated by experimental angular light scattering spectra of epithelial cells for scattering angles from 1.25 to 173.8 degrees and in the spectral range from 400nm to 700nm.