Bridging Structure and Process in Developmental Biology through New Imaging Technologies

Many unexpected discoveries in developmental biology have depended on advancement of imaging technologies to visualize developmental processes as they unfold across multiple spatial and temporal scales. This essay surveys the recent advances in imaging, highlighting emerging capabilities with an eye toward those poised to have the greatest impact on developmental biology

Plasma Membrane Polarity and Compartmentalization Are Established before Cellularization in the Fly Embryo

SummaryPatterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. We propose that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo

Active translocon complexes labeled with GFP–Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–Dad1 in the ER membranes, but to a level that is still lower than that of free GFP–Dad1. This suggests that GFP–Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains

ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells

Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with ∼40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly–disassembly cycle of the COPI coat in vivo

The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER

Peroxisomes are ubiquitous organelles that proliferate under different physiological conditions and can form de novo in cells that lack them. The endoplasmic reticulum (ER) has been shown to be the source of peroxisomes in yeast and plant cells. It remains unclear, however, whether the ER has a similar role in mammalian cells and whether peroxisome division or outgrowth from the ER maintains peroxisomes in growing cells. We use a new in cellula pulse-chase imaging protocol with photoactivatable GFP to investigate the mechanism underlying the biogenesis of mammalian peroxisomes. We provide direct evidence that peroxisomes can arise de novo from the ER in both normal and peroxisome-less mutant cells. We further show that PEX16 regulates this process by being cotranslationally inserted into the ER and serving to recruit other peroxisomal membrane proteins to membranes. Finally, we demonstrate that the increase in peroxisome number in growing wild-type cells results primarily from new peroxisomes derived from the ER rather than by division of preexisting peroxisomes