An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking.

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear.

ERK7 is a negative regulator of protein secretion in response to amino-acid starvation by modulating Sec16 membrane association.

RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic-Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. We found that ERK7 negatively regulates secretion in response to serum and amino-acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C-terminus of Sec16, a key ER exit site component.

Golgi bypass: skirting around the heart of classical secretion.

Classical secretion consists of the delivery of transmembrane and soluble proteins to the plasma membrane and the extracellular medium, respectively, and is mediated by the organelles of the secretory pathway, the Endoplasmic Reticulum (ER), the ER exit sites, and the Golgi, as described by the Nobel Prize winner George Palade (Palade 1975). At the center of this transport route, the Golgi stack has a major role in modifying, processing, sorting, and dispatching newly synthesized proteins to their final destinations. More recently, however, it has become clear that an increasing number of transmembrane proteins reach the plasma membrane unconventionally, either by exiting the ER in non-COPII vesicles or by bypassing the Golgi.

Identification of ER proteins involved in the functional organisation of the early secretory pathway in Drosophila cells by a targeted RNAi screen.

Background: In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation.

The multiple facets of the Golgi reassembly stacking proteins.

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo.

ISH-IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level.

This protocol describes the combination of in situ hybridization (ISH) with cryo-immunolabeling methods to allow the simultaneous detection at the ultrastructural level of mRNAs and proteins. The procedure consists of five steps and takes 4-5 d: (i) acquisition of ultrathin frozen sections of chemically fixed tissues or cells; (ii) hybridization of the sections with digoxigenin (DIG) or biotin-labeled RNA probes; (iii) detection of the bound probe with antibodies and protein A-gold (PAG); (iv) labeling of proteins of interest (optional); and (v) visualization by transmission electron microscopy (immuno-electron microscopy (IEM)). This technique allows the simultaneous detection of endogenous/overexpressed/injected RNAs and proteins while preserving the cell ultrastructure.

Distinguishing direct from indirect roles for bicoid mRNA localization factors.

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved.

The Golgi apparatus: lessons from Drosophila.

Historically, Drosophila has been a model organism for studying molecular and developmental biology leading to many important discoveries in this field. More recently, the fruit fly has started to be used to address cell biology issues including studies of the secretory pathway, and more specifically on the functional integrity of the Golgi apparatus. A number of advances have been made that are reviewed below.

Integrins mediate their unconventional, mechanical-stress-induced secretion via RhoA and PINCH in Drosophila.

During the epithelium remodelling such as the flattening of the Drosophila follicular epithelium, the alpha-integrin subunits are unconventionally secreted through a dGRASP-dependent route that is built de novo. The biogenetic process starts with the upregulation of a small subset of targeted mRNAs, including dgrasp. Here, we show that dgrasp mRNA upregulation is triggered by the tension of the underlying oocyte and by applied external forces at the basal side of the follicular epithelium.

The early secretory pathway in development: a tale of proteins and mRNAs.

The secretory pathway ensures the proper delivery of secreted proteins to the extracellular medium and of transmembrane proteins to almost all membrane cellular compartments. During their transport in the different compartments making up this pathway, newly synthesized proteins are modified and dispatched to their final destinations. So far, this pathway has mostly been studied in tissue cultured cells or yeast but recently, mutations in genes encoding key proteins of this pathway have been shown to lead to severe developmental defects in different model organisms.

dGRASP-mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling.

Integral plasma membrane proteins are typically transported in the secretory pathway from the endoplasmic reticulum and the Golgi complex. Here we show that at specific stages of Drosophila development corresponding to morphological changes in epithelia, apposed basolateral membranes separate slightly, allowing new plasma membrane contacts with basal extracellular matrix. At these sites, newly synthesized integrin alpha subunits are deposited via a mechanism that appears to bypass the Golgi.

Golgi ribbon unlinking: an organelle-based G2/M checkpoint.

Cell cycle checkpoints have been associated with signaling mechanisms monitoring the genetic material for abnormalities and inaccuracy in partitioning, such as DNA lesions or failure in chromosome attachment to kinetochores. However, it becomes more evident that other checkpoints are turned on by cytoplasmic events, such as the cytoskeleton organisation and the organelle structure. Here, we summarize recent evidence strongly suggesting that the integrity of the Golgi ribbon, and more precisely the tubules interconnecting the Golgi stacks to form this ribbon, at late G2/early prophase, is linked to a Golgi-related G2/M checkpoint.

The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE.

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. Here we show that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis.

dGRASP localization and function in the early exocytic pathway in Drosophila S2 cells.

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked.

mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes.

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-alpha synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. Here, we show, by (immuno)electron microscopy that the exocytic pathway in stage 9-10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units.