Endocytosis in Drosophila: progress, possibilities, prognostications.

By many outside the field, endocytosis is often perceived as a "house-keeping" function performed via identical mechanisms in yeast and man. Recent discoveries have done much to reduce this misperception. (1) Endocytosis occurs via different mechanisms and different pathways in different cellular contexts. (2) Molecular mechanisms that regulate homologous pathways in unicellular and multicellular organisms show considerable variance. (3) Temporally controlled endocytosis of specific regulatory molecules underlies several important and intricate biological processes including synapse formation, synaptic plasticity, cell fate determination, and morphogen gradient formation. Interactions between endocytosis and cytoskeletal and signaling pathways have been particularly revealing. In this intellectual context, Drosophila has become invaluable as a metazoan genetic model in which to understand the many faces of endocytosis. This review discusses two aspects of work in Drosophila: (a) its contributions toward understanding fundamental mechanisms that underlie the operation of endocytic pathways; (b) how analyses in Drosophila provide insights into varied biological processes regulated by endocytosis. In addition, while offering our commentary on merits and limitations of Drosophila work, we speculate on likely areas for contributions and future research on endocytosis in Drosophila.     

Functional dissection of a eukaryotic dicistronic gene: transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants

The dicistronic Drosophila stoned mRNA produces two proteins, stonedA and stonedB, that are localized at nerve terminals. While the stoned locus is required for synaptic-vesicle cycling in neurons, distinct or overlapping synaptic functions of stonedA and stonedB have not been clearly identified. Potential functions of stoned products in nonneuronal cells remain entirely unexplored in vivo. Transgene-based analyses presented here demonstrate that exclusively neuronal expression of a dicistronic stoned cDNA is sufficient for rescue of defects observed in lethal and viable stoned mutants. Significantly, expression of a monocistronic stonedB trangene is sufficient for rescuing various phenotypic deficits of stoned mutants, including those in organismal viability, evoked transmitter release, and synaptotagmin retrieval from the plasma membrane. In contrast, a stonedA transgene does not alleviate any stoned mutant phenotype. Novel phenotypic analyses demonstrate that, in addition to regulation of presynaptic function, stoned is required for regulating normal growth and morphology of the motor terminal; however, this developmental function is also provided by a stonedB transgene. Our data, although most consistent with a hypothesis in which stonedA is a dispensable protein, are limited by the absence of a true null allele for stoned due to partial restoration of presynaptic stonedA by transgenically provided stonedB. Careful analysis of the effects of the monocistronic transgenes together and in isolation clearly reveals that the presence of presynaptic stonedA is dependent on stonedB. Together, our findings improve understanding of the functional relationship between stonedA and stonedB and elaborate significantly on the in vivo functions of stonins, recently discovered phylogenetically conserved stonedB homologs that represent a new family of "orphan" medium (mu) chains of adaptor complexes involved in vesicle formation. Data presented here also provide new insight into potential mechanisms that underlie translation and evolution of the dicistronic stoned mRNA.     

A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation

Rapidly reversible, temperature-sensitive (ts) paralytic mutants of Drosophila have been useful in delineating immediate in vivo functions of molecules involved in synaptic transmission. Here we report isolation and characterization of orangi (org), an enhancer of shibire (shi), a ts paralytic mutant in Drosophila dynamin. org is an allele of the stress sensitive B (sesB) locus that encodes a mitochondrial adenine nucleotide translocase (ANT) and results in a unique ts paralytic behavior that is accompanied by a complete loss of synaptic transmission in the visual system. sesB(org) reduces the restrictive temperature for all shi(ts) alleles tested except for shi(ts1). This characteristic allele-specific interaction of sesB(org) with shi is shared by abnormal wing discs (awd), a gene encoding nucleoside diphosphate kinase (NDK). sesB(org) shows independent synergistic interactions, an observation that is consistent with a shared pathway by which org and awd influence shi function. Genetic and electrophysiological analyses presented here, together with the observation that the sesB(org) mutation reduces biochemically assayed ANT activity, suggest a model in which a continuous mitochondrial ANT-dependent supply of ATP is required to sustain NDK-dependent activation of presynaptic dynamin during a normal range of synaptic activity.     

Regulation of dynamin by nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is required for multiple cellular functions, including cell growth, motility, and differentiation, and its loss is associated with pathologies including tumor metastasis. A recent study has revealed a previously unknown function for NDP kinase as positive regulator of dynamin, a GTPase essential for endocytosis. In this review we describe the evidence that NDP kinase function is essential for endocytosis and also elaborate on a mechanism for NDP kinase regulation of dynamin. Recently documented interactions between endocytosis and cell signaling have revealed new insights into potential mechanisms of cancer. In this context, we discuss the possible relevance of NDP kinase and dynamin interaction for tumor suppression.     

Dispersal dynamics of SARS-CoV-2 lineages during the first epidemic wave in New York City

During the first phase of the COVID-19 epidemic, New York City rapidly became the epicenter of the pandemic in the United States. While molecular phylogenetic analyses have previously highlighted multiple introductions and a period of cryptic community transmission within New York City, little is known about the circulation of SARS-CoV-2 within and among its boroughs. We here perform phylogeographic investigations to gain insights into the circulation of viral lineages during the first months of the New York City outbreak. Our analyses describe the dispersal dynamics of viral lineages at the state and city levels, illustrating that peripheral samples likely correspond to distinct dispersal events originating from the main metropolitan city areas. In line with the high prevalence recorded in this area, our results highlight the relatively important role of the borough of Queens as a transmission hub associated with higher local circulation and dispersal of viral lineages toward the surrounding boroughs.     

Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca²⁺-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.     

An O(n log n)-time algorithm for the restriction scaffold assignment problem.

The restriction scaffold assignment problem takes as input two finite point sets S and T (with S containing more points than T ) and establishes a correspondence between points in S and points in T , such that each point in S maps to exactly one point in T and each point in T maps to at least one point in S. An algorithm is presented that finds a minimum-cost solution for this problem in O(n log n) time, provided that the points in S and T are restricted to lie on a line and the cost function delta is the L(1) metric. This algorithm runs in linear time, if S and T are presorted. This improves the previously best-known O(n (2))-time algorithm for this problem.     

Photoinduced destabilization of liposomes.

The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents.