Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-β/Smad and Wnt/β-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

The Ca2+ channel β2 subunit is selectively targeted to the axon terminals of supraoptic neurons

The assembly of high voltage-activated Ca²⁺ channels with different β subunits influences channel properties and possibly subcellular targeting. We studied β subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca_Vβ subunits (Ca_Vβ₁-Ca_Vβ₄) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 β subunits are expressed in both locations, but that Ca_Vβ₂ had the highest relative expression in the neurohypophysis. These data suggest that the Ca_Vβ₂ subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca²⁺ channels.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Immunotoxins up to the present day

Immunotoxins consist of monoclonal or polyclonal antibodies conjugated to bacterial or plant toxins. The toxins used are typically of the A-B type in which a toxic A chain is coupled to a B chain responsible for cell binding and facilitation of A chain entry into the cytosol. Two broad strategies have been followed: coupling intact toxins, or A chains alone, to antibodies. This review examines current progress inin vitro andin vivo research, including recent clinical studies, concentrating principally on ricin or ricin A chain conjugates. The future role of conjugates using membrane-acting toxins, immunolysins, is also discussed.