Detection of in situ cleaved p115 with the cut specific antibodies in rapid protein inactivation system by tobacco etch viral protease cleavage

Gene perturbation methods are commonly used in the study of gene and protein function. The authors of this paper recently developed a rapid protein inactivation technique utilizing tobacco etch virus (TEV)-derived protease. TEV protease recognizes the ENLYFQG (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) amino acid sequence and specifically cleaves between Q and G. The authors developed antibodies that recognize the cleaved TEV (ENLYFQ) sequence, both in vitro and in vivo, but do not bind to uncleaved TEV (ENLYFQG). Using these antibodies, in situ protein cleavage was successfully detected. These antibodies used in combination with the TEV protease may be a useful complement to other perturbation methods

The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus

Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications

Giantin Affects Golgi Stack Connection

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi

The Golgin Protein Giantin Regulates Interconnections Between Golgi Stacks

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking

Stage-Specific Effects of Ionizing Radiation during Early Development

Early embryonic cells are sensitive to genotoxic stressors such as ionizing radiation. However, sensitivity to these stressors varies depending on the embryonic stage. Recently, the sensitivity and response to ionizing radiation were found to differ during the preimplantation period. The cellular and molecular mechanisms underlying the change during this period are beginning to be elucidated. In this review, we focus on the changes in radio-sensitivity and responses to ionizing radiation during the early developmental stages of the preimplantation (before gastrulation) period in mammals, Xenopus, and fish. Furthermore, we discuss the underlying cellular and molecular mechanisms and the similarities and differences between species

Nerve independent limb induction in axolotls

AbstractUrodele amphibians can regenerate their limbs. During limb regeneration, dermal fibroblasts are transformed into undifferentiated cells called blastema cells. These dermis–blastema cells show multipotency. Such so-called endogenous reprogramming of cell differentiation is one of the main targets of amphibian limb regeneration studies. It is well recognized that nerve presence controls the initiation of limb regeneration. Accordingly, nerve factors have been sought in amphibian limb regeneration. To investigate it, a relatively new study system called the accessory limb model (ALM) was developed. Using ALM, two signaling cascades (Fgf and Gdf5 signaling) came under focus. In the present study, Growth and differentiation factor-5 (Gdf5) application to wounded skin initiated limb regeneration responses and resulted in induction of a blastema-like structure in the absence of a nerve. However, the Gdf5-induced structure showed defects as a regeneration blastema, such as absence of detectable Prrx1 expression by in situ hybridization. The defects could be remedied by additional Fibroblasts growth factor (Fgf) inputs. These two inputs (Gdf5 and Fgfs) were sufficient to substitute for the nerve functions in the induction of limb regeneration. Indeed, Fgf2, Fgf8, and Gdf5 applications with the contralateral skin graft resulted in limb formation without nerve supply. Furthermore, acquisition of cartilage differentiation potential of dermal fibroblasts was tested in an in vivo and in vitro combination assay. Dermal fibroblasts cultured with Gdf5 were difficult to participate in cartilage formation when the cultured cells were grafted into cartilage forming region. In contrast, dermal fibroblasts cultured with Fgf2 and Fgf8 became easier to participate into cartilage formation in the same procedure. These results contribute to our understanding of molecular mechanisms of the early phase of amphibian limb regeneration

Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut

The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activation-induced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID(–/–) mice. Reconstitution of lamina propria IgA production in AID(–/–) mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system