We identified Xenopus pericentriolar material-1 (PCM-1), which had been reported to constitute pericentriolar material, cloned its cDNA, and generated a specific pAb against this molecule. Immunolabeling revealed that PCM-1 was not a pericentriolar material protein, but a specific component of centriolar satellites, morphologically characterized as electron-dense granules, ∼70–100 nm in diameter, scattered around centrosomes. Using a GFP fusion protein with PCM-1, we found that PCM-1–containing centriolar satellites moved along microtubules toward their minus ends, i.e., toward centrosomes, in live cells, as well as in vitro reconstituted asters. These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization. Next, to understand the relationship between centriolar satellites and centriolar replication, we examined the expression and subcellular localization of PCM-1 in ciliated epithelial cells during ciliogenesis. When ciliogenesis was induced in mouse nasal respiratory epithelial cells, PCM-1 immunofluorescence was markedly elevated at the apical cytoplasm. At the electron microscopic level, anti–PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis. These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.

Gamma-tubulin regulates the nucleation of microtubules, but knowledge of its functions in vivo is still fragmentary. Here, we report the identification of two closely related gamma-tubulin isoforms, TUBG1 and TUBG2, in mice, and the generation of TUBG1- and TUBG2-deficient mice. TUBG1 was expressed ubiquitously, whereas TUBG2 was primarily detected in the brain. The development of TUBG1-deficient (Tubg1-/-) embryos stopped at the morula/blastocyst stages due to a characteristic mitotic arrest: the mitotic spindle was highly disorganized, and disorganized spindles showed one or two pole-like foci of bundled MTs that were surrounded by condensed chromosomes. TUBG2 was expressed in blastocysts, but could not rescue the TUBG1 deficiency. By contrast, TUBG2-deficient (Tubg2-/-) mice were born, grew, and intercrossed normally. In the brain of wild-type mice, TUBG2 was expressed in approximately the same amount as TUBG1, but no histological abnormalities were found in the Tubg2-/- brain. These findings indicated that TUBG1 and TUBG2 are not functionally equivalent in vivo, that TUBG1 corresponds to conventional gamma-tubulin, and that TUBG2 may have some unidentified function in the brain.

We have developed a mass microscope (mass spectrometry imager with spatial resolution higher than the naked eye) equipped with an atmospheric pressure ion-source chamber for laser desorption/ionization (AP-LDI) and a quadrupole ion trap time-of-flight (QIT-TOF) analyzer. The optical microscope combined with the mass spectrometer permitted us to precisely determine the relevant tissue region prior to performing imaging mass spectrometry (IMS). An ultraviolet laser tightly focused with a triplet lens was used to achieve high spatial resolution. An atmospheric pressure ion-source chamber enables us to analyze fresh samples with minimal loss of intrinsic water or volatile compounds. Mass-microscopic AP-LDI imaging of freshly cut ginger rhizome sections revealed that 6-gingerol ([M + K](+)at m/z 333.15, positive mode; [M - H](-) at m/z 293.17, negative mode) and the monoterpene ([M + K](+) at m/z 191.09), which are the compounds related to pungency and flavor, respectively, were localized in oil drop-containing organelles. AP-LDI-tandem MS/MS analyses were applied to compare authentic signals from freshly cut ginger directly with the standard reagent. Thus, our atmosphere-imaging mass spectrometer enabled us to monitor a quality of plants at the organelle level.

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein-lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport. INTRODUCTIONMotile cilia and flagella are membrane-bound, microtubulecontaining cell surface projections that are differentiated by their length, movement characteristics, and numbers per cell (Haimo and Rosenbaum, 1981;Mitchell, 2007;Satir and Christensen, 2007). Flagella-containing cells generally have only one or two flagella, which are long motile structures that beat independently and exhibit an undulating motion. Conversely, cilia are generally much more numerous and are shorter motile projections with an oscillating to-and-fro motion. In humans, flagella are observed only in sperm cells, whereas motile cilia are observed on the respiratory epithelium, along the female reproductive tract, and on the ependymal cells lining the ventricles of the brain.Early studies using electron microscopy showed that the terminal end of mammalian motile cilia is quite distinct from that of sperm flagella (see Figure 6). At the terminal end of mammalian sperm flagella, peripheral doublet microtubules lose the outer B subfibers or separate into two singlet microtubules. Each singlet microtubule approaches the cell membrane, becomes electron dense and terminates successively. Only a few peripheral singlet subfibers continue to the tip of the sperm tail (Woolley and Nickels, 1985;Afzelius et al., 1995;Suzuki and Nagano, 2002). In contrast, the apical structure of mammalian cilia has a distinct fine structure at the termination site of the axonemal microtubules. The outer B subfibers of peripheral doublet microtubules terminate at the distal portion of cilia, and only the singlet A subfibers continue to the tip. T...

Imaging mass spectrometry (IMS) is a powerful tool for detecting and visualizing biomolecules in tissue sections. The technology has been applied to several fields, and many researchers have started to apply it to pathological samples. However, it is very difficult for inexperienced users to extract meaningful signals from enormous IMS datasets, and the procedure is time-consuming. We have developed software, called IMS Convolution with regions of interest (ROI), to automatically extract meaningful signals from IMS datasets. The processing is based on the detection of common peaks within the ordered area in the IMS dataset. In this study, the IMS dataset from a mouse eyeball section was acquired by a mass microscope that we recently developed, and the peaks extracted by manual and automatic procedures were compared. The manual procedure extracted 16 peaks with higher intensity in mass spectra averaged in whole measurement points. On the other hand, the automatic procedure using IMS Convolution easily and equally extracted peaks without any effort. Moreover, the use of ROIs with IMS Convolution enabled us to extract the peak on each ROI area, and all of the 16 ion images on mouse eyeball tissue were from phosphatidylcholine species. Therefore, we believe that IMS Convolution with ROIs could automatically extract the meaningful peaks from large-volume IMS datasets for inexperienced users as well as for researchers who have performed the analysis.

Imaging mass spectrometry (IMS) is a molecular imaging technique for various molecules such as compounds, metabolites and proteins to visualize spatial distribution of these components in a tissue sample. In recent years, the measurement of the spatial distribution of components using IMS technique is being carried out intensively in many fields including pathology and drug discovery. Although the importance of IMS has been recognized widely, there are not so many efficient software programs to handle these kinds of data because of the difficulty in treating huge amount of spectra data from many spots, which sometimes amounts to tens of thousands. In this presentation we introduce new software we developed and report its performance. In order to correspond to various purposes, the software was made to consist of four programs as shown below. They achieve quick, automatic, and comprehensive analyses of the important peaks for the first time in the world. To detect important peaks of bio-molecules in each spectrum from mass spectrometry, we first developed a high-speed program for peak picking using a common peak method for IMS data, named IMS Convolution (IMSC). Once some regions of interests (ROI) such as cancer, interstitial and normal regions in a tissue sample are specified through graphical interface program, common peaks in each ROI are automatically detected by IMS convolution. Users can modify a definition of a threshold of common peaks easily, and the IMSC software picks up revised common peaks very quickly. Furthermore, we developed three more programs, named Spatial Peak Detectors (SPeaD) -1, 2 and 3, which use peak picking results by the IMSC. SPeaD-1 is for detecting a small set of peaks and it can discriminate two or more ROIs based on a method from machine learning theory. SPeaD-2 is for detecting cell specific peaks without ROI information. Cancer specific small molecules can be detected when cancer cells are distributed individually in normal tissue. SPeaD-3 is for detecting region specific peaks without ROI information. SPeaD-3 picks up all peaks of the same mass expressed in a clustered region defined by sets of adjacent spots. In evaluating the performance of the software, we used quasi-samples for IMSC and SPeaD-1 and cancer tissue samples for SPeaD-3. IMSC detects all the peaks beyond the noise level. Computation time is within 10 minutes for the data of ANALYZE format file size about 2.5GB with 62,500 (= 250 x 250) spots and with the range of m/z 650-1500. A workstation used for the analysis is equipped with Xeon E5504 CPU (2.0GHz) and 3GB memory. For SPeaD programs, computation time depends on the number of spots and m/z range but falls within about 30 minutes. Furthermore, we analyzed IMS data for hepatic micrometastasis of human colon cancer xenografts in superimmunodeficient NOG mice. Some specific bio-molecules of hepatic micrometastasis were detected quickly and automatically. Further details are given in the presentation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3963. doi:1538-7445.AM2012-3963