Processing and Distribution of dec-1 Eggshell Products in Drosophila Melanogaster

Understanding the molecular mechanisms that underlie generation of complex three-dimensional structures is one of the most fascinating and challenging problems in developmental biology. The Drosophila eggshell formation represents a favorable model to study the assembly of suprarnolecular structure during development in a genetically amenable eukaryote. Among the various eggshell genes cloned, dec-1 (defective chorion) offers a good opportunity to analyze the role of proteolysis in the in vivo regulation of an assembly pathway.· The dec-1 locus generates three proteins of 106 kDa (fcl06), 125 kDa (fcl25), and 177 kDa (fcl77). Previous studies established that fcl06 is first processed into a protein of about 80 kDa, which in turn, is cleaved into a c-terminal derivative of approximately 60 kDa (s60). Preliminary observations indicated that fcl25 may also be processed during oogenesis. Polyclonal antibodies produced against trpE-fusion proteins containing selected regions of dec-1 open reading frame were used to further analyze the proteolytic processing of .the dec-1 proteins as well as their in situ distribution in the eggshell. It was found that fcl06, fcl25 and fcl77 follow different regulated maturation pathways that result in the production of several stable proteins with distinctive N- and C-termini. Each one of the dec-1 mature proteins shows a different distribution in the eggshell. fcl06 processing generates three final products, the previously identified s60, a 25 kDa (s25) and a 20 kDa (s20) proteins. s60, fractionates between the vitelline membrane (VM) and the chorion, the two main layers of the eggshell; s25 is a chorion component while s20 localizes into vesicles inside the oocyte. fc125 processing yields a stable protein of approximately 95 kDa (s95) which during early choriogenesis is observed in the VM and in the forming chorion. The final processing derivative of fc177 localizes in the mature eggshell, inside rounded cavities characteristic of the shell chorion. The different behavior in the egg chamber of the various mature dec-1 proteins indicates that there is no functional redundancy among them. The complexity of the dec-1 locus suggests that the assembly of the eggshell is a highly regulated process

Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.

1. Intracellular Cl- activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled Cl- -selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was -57.7 +/- 1.0 mV and aiCl was 23.6 +/- 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min-1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl when external Cl- was removed were too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl- was dependent on the extracellular Cl- activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half-maximal activation of about 50 mM-external Cl-. 4. The steady-state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl- after intracellular Cl- depletion was abolished in the absence of either Na+ or K+ in the bathing solution. 5. The reaccumulation of Cl- was inhibited by furosemide (0.5-1 x 10(-3) M) or bumetanide (10(-5) M). The decrease in aiCl observed in Cl- -free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl- -free solutions to about 75% their initial volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in DRG neurones. The mechanism of Cl- transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+:K+:Cl- co-transport system mediates the active transport of Cl- across the cell membrane of DRG neurones

Intracellular Chloride Regulation in Amphibian Dorsal Root Ganglion Neurones Studied With Ion-Selective Microelectrodes

1. Intracellular Cl- activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled Cl- -selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was -57.7 +/- 1.0 mV and aiCl was 23.6 +/- 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min-1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl when external Cl- was removed were too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl- was dependent on the extracellular Cl- activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half-maximal activation of about 50 mM-external Cl-. 4. The steady-state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl- after intracellular Cl- depletion was abolished in the absence of either Na+ or K+ in the bathing solution. 5. The reaccumulation of Cl- was inhibited by furosemide (0.5-1 x 10(-3) M) or bumetanide (10(-5) M). The decrease in aiCl observed in Cl- -free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl- -free solutions to about 75% their initial volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in DRG neurones. The mechanism of Cl- transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+:K+:Cl- co-transport system mediates the active transport of Cl- across the cell membrane of DRG neurones