Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.

Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.

Electron Microscopy Imaging of Zinc Soaps Nucleation in Oil Paint.

Using the recently developed techniques of electron tomography, we have explored the first stages of disfiguring formation of zinc soaps in modern oil paintings. The formation of complexes of zinc ions with fatty acids in paint layers is a major threat to the stability and appearance of many late 19th and early 20th century oil paintings. Moreover, the occurrence of zinc soaps in oil paintings leading to defects is disturbingly common, but the chemical reactions and migration mechanisms leading to large zinc soap aggregates or zones remain poorly understood. State-of-the-art scanning (SEM) and transmission (TEM) electron microscopy techniques, primarily developed for biological specimens, have enabled us to visualize the earliest stages of crystalline zinc soap growth in a reconstructed zinc white (ZnO) oil paint sample. In situ sectioning techniques and sequential imaging within the SEM allowed three-dimensional tomographic reconstruction of sample morphology. Improvements in the detection and discrimination of backscattered electrons enabled us to identify local precipitation processes with small atomic number contrast. The SEM images were correlated to low-dose and high-sensitivity TEM images, with high-resolution tomography providing unprecedented insight into the structure of nucleating zinc soaps at the molecular level. The correlative approach applied here to study phase separation, and crystallization processes specific to a problem in art conservation creates possibilities for visualization of phase formation in a wide range of soft materials.

Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery.

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.

Structural insights into the organization of the cavin membrane coat complex.

Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.

Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis of insulin granules.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from ß-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking ß-cell ABCA1. RESEARCH DESIGN AND METHODS: Calcium imaging, patch clamp, and membrane capacitance were used to assess the effect of ABCA1 deficiency on calcium flux, ion channel function, and exocytosis in islet cells. Electron microscopy was used to analyze ß-cell ultrastructure. The quantity and distribution of proteins involved in insulin-granule exocytosis were also investigated. RESULTS: We show that a lack of ß-cell ABCA1 results in impaired depolarization-induced exocytotic fusion of insulin granules. We observed disturbances in membrane microdomain organization and Golgi and insulin granule morphology in ß-cells as well as elevated fasting plasma proinsulin levels in mice in the absence of ß-cell ABCA1. Acute cholesterol depletion rescued the exocytotic defect in ß-cells lacking ABCA1, indicating that elevated islet cholesterol accumulation directly impairs granule fusion and insulin secretion. CONCLUSIONS: Our data highlight a crucial role of ABCA1 and cellular cholesterol in ß-cells that is necessary for regulated insulin granule fusion events. These data suggest that abnormalities of cholesterol metabolism may contribute to the impaired ß-cell function in diabetes.

Working with oocyte nuclei: cytological preparations of active chromatin and nuclear bodies from amphibian germinal vesicles.

The giant nucleus or germinal vesicle (GV) of amphibian oocytes presents a remarkable opportunity to examine nuclear structures in unprecedented levels of detail. By making use of spread preparations of GVs, it is possible to investigate the structure and function of transcription units in active chromatin and a variety of nuclear bodies, all within the limits of resolution of the light microscope. The basic method for producing GV spreads that is described here is based on simple manual dissection and, therefore, it permits the preparation of nuclear components that have suffered a minimum of experimental manipulation. The particular method described is based on the use of oocytes from a salamander, the axolotl, although the approach is robust and applicable with minor modification to two other model amphibian species, Xenopus laevis and X. tropicalis. One common approach to investigating the molecular organisation of oocyte nuclear structures by immunofluorescent staining of endogenous or exogenous polypeptides is also described.

Patterns of lymph node spread of cutaneous squamous cell carcinoma of the head and neck.

BACKGROUND: Among patients with cutaneous squamous cell carcinoma (SCC) of the head and neck, recent studies have shown that those with involvement of the parotid gland also have a high incidence of neck node involvement. Treatment of the neck by either surgery or radiotherapy is therefore recommended among patients with parotid SCC, even if clinical examination is negative. The aim of this study was first to analyze patterns of metastatic spread in the parotid and cervical lymph nodes and then to correlate the pattern of involved nodes with the primary cutaneous site in order to guide the appropriate extent of surgery, should neck dissection be used to treat the neck in patients with parotid SCC. METHODS: A cohort of 209 patients with cutaneous SCC of the head and neck and clinically evident regional metastatic disease was reviewed retrospectively from 3 Australian institutions. The distribution of involved nodes was obtained from pathology reports; the anatomic sites of primary cutaneous cancers were then correlated with these findings. RESULTS: Among 209 patients, 171 (82%) had clinical parotid involvement. Of these, 28 had clinical neck disease, whereas 143 had parotid disease alone. Thirty-eight (18%) patients had neck disease only. A total of 199 patients were treated surgically, whereas 10 received radiotherapy alone. Surgery included 172 parotidectomies and 151 neck dissections (93 of which were elective). Primary sites were cheek (21.7%), pinna (20.4%), temple (15.8%), forehead (15.8%), postauricular region (5.9%), neck (5.3%), anterior scalp (5.3%), posterior scalp (3.3%), periorbital (3.3%), nose (2.6%), and chin (0.6%). Among pathologically positive necks, level II was most frequently involved (79%). Level IV (13%) and level V (17%) were only involved in extensive lymph node disease, the exception being for isolated level V metastases from the posterior scalp. CONCLUSIONS: Primary sites were mainly localized to the lateral aspect of the head. Among patients with cutaneous SCC involving the parotid and neck, level II was the most commonly involved neck level. The distribution of involved nodes suggests that in a patient with parotid involvement and a clinically negative neck with an anterolateral primary, a supraomohyoid neck dissection, always including the external jugular lymph node(s) would be appropriate. In the case of a posterior primary, level V should be dissected as well. In patients with parotid SCC and a clinically positive neck, a comprehensive neck dissection is recommended.

Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins.

In yeast, separation of duplicated spindle pole bodies (SPBs) (centrosomes in higher eukaryotes) is an indispensable step in the assembly of mitotic spindle and is triggered by severing of the bridge that connects the sister SPBs. This process requires Cdk1 (Cdc28) activation by Tyrosine 19 dephosphorylation. We show that cells that fail to activate Cdk1 are devoid of spindles due to persistently active APCCdh1, which targets microtubule-associated proteins Cin8, Kip1 and Ase1 for degradation. Tyrosine 19 dephosphorylation of Cdk1 is necessary to specifically prevent proteolysis of these proteins. Interestingly, SPB separation is dependent on the microtubule-bundling activity of Cin8 but not on its motor function. Since ectopic expression of proteolysis-resistant Cin8, Kip1 or Ase1 is sufficient for SPB separation even in the absence of Cdc28-Clb activity, we suggest that stabilization of these mechanical force-generating proteins is the predominant role of Cdc28-Clb in centrosome separation.

Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles.

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.