Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.     

Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage

Transgenic mice carrying the diphtheria toxin A gene driven by mouse gamma 2-crystallin promoter sequences manifest microphthalmia due to ablation of fiber cells in the ocular lens. Here we map ablation events in the lens by crossing animals hemizygous for the ablation construct with transgenic mice homozygous for the in situ lacZ reporter gene driven by identical gamma 2-crystallin promoter sequences. By comparing the spatial distribution of lacZ-expressing cells and the profile of gamma-crystallin gene expression in the lenses of normal and microphthalmic offspring, the contributions of specific cell types to lens development were examined. The results suggest that phenotypically and developmentally distinct populations of lens fiber cells are able to contribute to the lens nucleus during organogenesis. We also show that dosage of the transgene and its site of integration influence the extent of ablation. In those mice homozygous for the transgene and completely lacking cells of the lens lineage, we show that the sclera, cornea, and ciliary epithelium are reduced in size but, otherwise, reasonably well formed. In contrast, the anterior chamber, iris, and vitreous body are not discernible while the sensory retina is highly convoluted and extensively fills the vitreous chamber.     

The ultrastructural basis for the identification of cell types in the pancreatic islets I. Guinea pig

Summary The pancreatic islets of the guinea pig have been studied by light and electron microscopy. The B granules in glutaraldehyde-fixed tissue often are cup-shaped with an indentation visible on one side of the granule. Phosphotungstic acid hematoxylin (PTAH) positive cells have been characterized by electron microscopy as three subtypes based on the size of the secretory granules. A_a cells are the most common and have secretory granules around 200 m in diameter. A_b cells have large secretory granules around 300 m in diameter and are relatively infrequent. A_c cells are the least common and have small (160 m) granules. Characteristic D cells are identifiable by electron microscopy and, on the basis of the subsequent study (Munger, Caramia, and Lacy, 1965), are identified as the silver positive cells observed by light microscopy.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases.-The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donohue.     

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.