Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster

Summary This study demonstrates that endocytosis in the oocyte of Drosophila melanogaster is reversibly blocked at the stage of pit formation by the temperature-sensitive, single-gene mutant, shibire ^ts1. Uptake of horseradish peroxidase conjugated with wheat-germ agglutinin was observed to be normal in mutant oocytes at 19°C, but was blocked at 29°C. After 10 min at 29°C, there was a build-up of coated pits along invaginations of the plasma membrane. Also, the endosomal compartment consisting of tubules, bulbs, and small yolk spheres, disappeared. Lowering the temperature to 19°C after 10 min at 29°C released a synchronized wave of endocytosis into a cytoplasm cleared of uptake-related organelles. By observing this synchronized wave after exposure to 19°C for varying durations, we determined that endocytosis proceeds as follows: coated pits/vesiclestubulessmall yolk spheresmature yolk spheres. The observations suggest that these organelles transform one into another within this sequence.     

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [¹⁴C]glycerol 3-phosphate and [¹⁴C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C₁₈-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.Abbreviation BSA bovine serum albumin     

Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.     

Fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles

Ca²⁺-induced transformation of phosphatidylcholine-phosphatidic acid vesicles to larger bilayer structures has been examined using nuclear magnetic resonance, electron microscopy, gel permeation and radioisotope tracer techniques. For concentrated vesicle preparations where phosphatidic acid content remains less than 50% of total lipid, transformation to larger well defined unilamellar structures can be induced. The size of the product formed is dependent on phosphatidic acid content and on Ca²⁺ content when Ca²⁺ levels are between 0.3 and 1.0 mol ratios with respect to phosphatidic acid. During transformation bilayer composition remains unchanged and internal contents are retained in the final structure. These properties are indicative of concerted two vesicle and multiple vesicle fusions. The controllable and concerted fusions make the phosphatidic acid system suitable for further mechanistic studies.     

Rab35: GEFs,GAPs and Effectors

Rabs are the largest family of small GTPases and are master regulators of membrane trafficking. Following activation by guanine‐nucleotide exchange factors (GEFs), each Rab binds a specific set of effector proteins that mediate the various downstream functions of that Rab. Then, with the help of GTPase‐activating proteins, the Rab converts GTP to GDP, terminating its function. There are over 60 Rabs in humans and only a subset has been analyzed in any detail. Recently, Rab35 has emerged as a key regulator of cargo recycling at endosomes, with an additional role in regulation of the actin cytoskeleton. Here, we will focus on the regulation of Rab35 activity by the connecdenn/DENND1 family of GEFs and the TBC1D10/EPI64 family of GTPase‐activating proteins. We will describe how analysis of these proteins, as well as a plethora of Rab35 effectors has provided insights into Rab35 function. Finally, we will describe how Rab35 provides a novel link between the Rab and Arf family of GTPases with implications for tumor formation and invasiveness .      

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

Time course of meiotic progression after transferring primary spermatocyte into ooplasm at different stages

This study attempted to investigate the time course of meiotic progression after transferring primary spermatocyte (PS) into ooplasm at different maturing stages. In present experiments, PSs were introduced into maturing ooplasts or oocytes by electrofusion. Higher fusion rate was obtained by phytohemagglutinin (PHA) agglutination than by perivitelline space (PVS) insertion. When the ooplasms prepared at 0, 2, 5, and 8.5 hr of in vitro maturation (IVM) were used as recipients and PSs were used as donors, the reconstructed cells extruded the first polar body (PB1) approximately 8.5, 7, 5.5, and 3 hr after electrofusion, respectively. Especially, when ooplasm cultured for 8.5 hr in vitro after GV removal was fused with PS, the PB1 was emitted 7-11 hr after electrofusion. Additionally, the PB1 extrusions of GV and pro-MI oocytes fertilized with PSs were 2.5 hr earlier than control oocytes. The results suggest that (1) PSs undergo the first meiosis in different time courses when introduced into ooplasm at different maturing stages; (2) GV material plays an important role in determining the timing of PB1 extrusion; and (3) first meiotic division of GV and pro-MI oocytes can be accelerated by introducing PS.     

An artificial promoter construct for heat-inducible misexpression during fish embryogenesis

Beside spatial distribution, timing of gene expression is a key parameter controlling gene function during embryonic development. Gain-of-function experiments can therefore have quite opposing results, depending on the time of gene activation. Induction techniques are necessary to control timing in these experiments from outside of the organism. Natural heat shock promoters constitute a simple inducible misexpression system, the main disadvantage is a high background level of expression. We present here a new heat stress-inducible bidirectional promoter consisting of multimerized heat shock elements (HSE). The simplified architecture of this promoter largely improves the properties needed for an efficient induction system: dramatically reduced background activity, improved inducibility, and loss of all tissue specific components. Based on this new artificial promoter, we present a transient induction system for fish embryos. Application of this new induction system for Fgf8 misexpression during embryonic development reveals different windows of competence during eye development. A dramatic early phenotype resulting in loss of the eyes is observed for conventional mRNA injection. Later activation, by using our inducible promoter, uncovers different eye phenotypes like cyclopic eyes. Even after 14 days, an efficient heat stress response could be evoked in the injected embryos. The HSE promoter therefore represents a new artificial heat shock promoter with superior properties, making possible transient experiments with inducible misexpression at various stages of development.