PLACENTAL STEREOLOGY: SPANNING THE LEVELS FROM MOLECULE TO WHOLE ORGAN

Stereology can provide hard (functionally-relevant) quantitative information at different levels of 3D structural organization. My researches have applied established methods to study organ function and developed novel methods to study subcellular localization of marker probes. We have studied whole-organ function (passive diffusion) in the human placenta and quantified various processes at tissue and subcellular levels and in normal and complicated pregnancies. Fetoplacental angiogenesis and villous growth and maturation involve phased changes particularly around mid-gestation. Growth is associated with increased numbers of cells or nuclei and counts have shown that villous trophoblast continuously renews itself via cytotrophoblast (CT) proliferation and recruitment and syncytiotrophoblast (ST) differentiation and extrusion. Integration of these processes results in changes in total oxygen (O2) diffusive conductance which match the growing fetal mass. Similar processes occur during development of the mouse placenta and at least some are compromised in human pregnancies. For example: [a] in pure pre-eclampsia (PE), villous and fetoplacental vascular volumes and surfaces are similar to those seen in uncomplicated pregnancies but reduced in pure intrauterine growth restriction (IUGR) and in PE+IUGR; [b] trophoblast extrusion is accelerated in PE and IUGR but the latter exhibits reduced CT proliferation and this perturbed steady state leads to smaller trophoblast volumes and surfaces; [c] O2 diffusive conductances alter in various pregnancy complications, including IUGR and PE+IUGR; [d] fetal weight is diminished, but diffusive transport increases, in placentas from mice exposed to urban air pollution. Finally, innovations in quantitative immunoelectron microscopy (immunoEM) have produced a portfolio of methods for revealing non-random distributions of marker gold particles in different cellular compartments and for testing whether patterns shift following experimental manipulation. Recently, the methods have been extended to study tissue and cellular distributions of nanoparticles.

CD317/Tetherin Is Enriched in the HIV-1 Envelope and Downregulated from the Plasma Membrane upon Virus Infection

ABSTRACT CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.

The m-AAA Protease Processes Cytochrome c Peroxidase Preferentially at the Inner Boundary Membrane of Mitochondria

The m-AAA protease is a conserved hetero-oligomeric complex in the inner membrane of mitochondria. Recent evidence suggests a compartmentalization of the contiguous mitochondrial inner membrane into an inner boundary membrane (IBM) and a cristae membrane (CM). However, little is known about the functional differences of these subdomains. We have analyzed the localizations of the m-AAA protease and its substrate cytochrome c peroxidase (Ccp1) within yeast mitochondria using live cell fluorescence microscopy and quantitative immunoelectron microscopy. We find that the m-AAA protease is preferentially localized in the IBM. Likewise, the membrane-anchored precursor form of Ccp1 accumulates in the IBM of mitochondria lacking a functional m-AAA protease. Only upon proteolytic cleavage the mature form mCcp1 moves into the cristae space. These findings suggest that protein quality control and proteolytic activation exerted by the m-AAA protease take place preferentially in the IBM pointing to significant functional differences between the IBM and the CM.

Taking apart the endocytic machinery

The formation of clathrin-coated endocytic vesicles is driven by a complex and highly dynamic molecular machinery. In this issue, Idrissi et al. (Idrissi, F.-Z., H. Grötsch, I.M. Fernández-Golbano, C. Presciatto-Baschong, H. Riezman, and M.-I. Geli. 2008. J. Cell Biol. 180:1219–1232) reveal some of the secrets of this machinery by analyzing the localizations of nine endocytic proteins during vesicle budding in yeast using quantitative immunoelectron microscopy.

Endocytosis and Sorting of ErbB2 and the Site of Action of Cancer Therapeutics Trastuzumab and Geldanamycin

ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.