Distinct Morphological Phenotypes of Cell Fusion Mutants

Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Δ fus2Δ, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus− zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

Formation and Turnover of NSF- and SNAP-containing “Fusion” Complexes Occur on Undocked, Clathrin-coated Vesicle–derived Membranes

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle–derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

Vacuole Fusion Regulated by Protein Phosphatase 2C in Fission Yeast

The gene ptc4+ encodes one of four type 2C protein phosphatases (PP2C) in the fission yeast Schizosaccharomyces pombe. Deletion of ptc4+ is not lethal; however, Δptc4 cells grow slowly in defined minimal medium and undergo premature growth arrest in response to nitrogen starvation. Interestingly, Δptc4 cells are unable to fuse vacuoles in response to hypotonic stress or nutrient starvation. Conversely, Ptc4 overexpression appears to induce vacuole fusion. These findings reveal a hitherto unrecognized function of type 2C protein phosphatases: regulation of vacuole fusion. Ptc4 localizes in vacuole membranes, which suggests that Ptc4 regulates vacuole fusion by dephosphorylation of one or more proteins in the vacuole membrane. Vacuole function is required for the process of autophagy that is induced by nutrient starvation; thus, the vacuole defect of Δptc4 cells might explain why these cells undergo premature growth arrest in response to nitrogen starvation.

A Specific Point Mutant at Position 1 of the Influenza Hemagglutinin Fusion Peptide Displays a Hemifusion Phenotype

We showed previously that substitution of the first residue of the influenza hemagglutinin (HA) fusion peptide Gly1 with Glu abolishes fusion activity. In the present study we asked whether this striking phenotype was due to the charge or side-chain volume of the substituted Glu. To do this we generated and characterized six mutants with substitutions at position 1: Gly1 to Ala, Ser, Val, Glu, Gln, or Lys. We found the following. All mutants were expressed at the cell surface, could be cleaved from the precursor (HA0) to the fusion permissive form (HA1-S-S-HA2), bound antibodies against the major antigenic site, bound red blood cells, and changed conformation at low pH. Only Gly, Ala, and Ser supported lipid mixing during fusion with red blood cells. Only Gly and Ala supported content mixing. Ser HA, therefore, displayed a hemifusion phenotype. The hemifusion phenotype of Ser HA was confirmed by electrophysiological studies. Our findings indicate that the first residue of the HA fusion peptide must be small (e.g., Gly, Ala, or Ser) to promote lipid mixing and must be small and apolar (e.g., Gly or Ala) to support both lipid and content mixing. The finding that Val HA displays no fusion activity underscores the idea that hydrophobicity is not the sole factor dictating fusion peptide function. The surprising finding that Ser HA displays hemifusion suggests that the HA ectodomain functions not only in the first stage of fusion, lipid mixing, but also, either directly or indirectly, in the second stage of fusion, content mixing.

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 × 10−6 M. Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca2+. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior.

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity

It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G1/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

A fusion pore phenotype in mast cells of the ruby-eye mouse

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse (ru/ru) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 ± 0.02 (wild type) to 0.65 ± 0.02 (ru/ru), and the average duration of these events increased from 418 ± 32 ms (wild type) to 1207 ± 89 ms (ru/ru). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.

The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGFβR kinase-dependent signaling pathways

The TEL/PDGFβR fusion protein is the product of the t(5;12) translocation in patients with chronic myelomonocytic leukemia. The TEL/PDGFβR is an unusual fusion of a putative transcription factor, TEL, to a receptor tyrosine kinase. The translocation fuses the amino terminus of TEL, containing the helix-loop-helix (HLH) domain, to the transmembrane and cytoplasmic domain of the PDGFβR. We hypothesized that TEL/PDGFβR self-association, mediated by the HLH domain of TEL, would lead to constitutive activation of the PDGFβR tyrosine kinase domain and cellular transformation. Analysis of in vitro-translated TEL/PDGFβR confirmed that the protein self-associated and that self-association was abrogated by deletion of 51 aa within the TEL HLH domain. In vivo, TEL/PDGFβR was detected as a 100-kDa protein that was constitutively phosphorylated on tyrosine and transformed the murine hematopoietic cell line Ba/F3 to interleukin 3 growth factor independence. Transformation of Ba/F3 cells required the HLH domain of TEL and the kinase activity of the PDGFβR portion of the fusion protein. Immunoblotting demonstrated that TEL/PDGFβR associated with multiple signaling molecules known to associate with the activated PDGFβR, including phospholipase C γ1, SHP2, and phosphoinositol-3-kinase. TEL/PDGFβR is a novel transforming protein that self-associates and activates PDGFβR-dependent signaling pathways. Oligomerization of TEL/PDGFβR that is dependent on the TEL HLH domain provides further evidence that the HLH domain, highly conserved among ETS family members, is a self-association motif.

Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic–nuclear transport

The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.

Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positive papillary renal cell carcinomas

The (X;1)(p11;q21) translocation is a recurrent chromosomal abnormality in a subset of human papillary renal cell carcinomas, and is sometimes the sole cytogenetic abnormality present. Via positional cloning, we were able to identify the genes involved. The translocation results in a fusion of the transcription factor TFE3 gene on the X chromosome to a novel gene, designated PRCC, on chromosome 1. Through this fusion, reciprocal translocation products are formed, which are both expressed in papillary renal cell carcinomas. PRCC is ubiquitously expressed in normal adult and fetal tissues and encodes a putative protein of 491 aa with a relatively high content of prolines. No relevant homologies with known sequences at either the DNA or the protein level were found.

Human AML1/MDS1/EVI1 fusion protein induces an acute myelogenous leukemia (AML) in mice: A model for human AML

The human t(3;21)(q26;q22) translocation is found as a secondary mutation in some cases of chronic myelogenous leukemia during the blast phase and in therapy-related myelodysplasia and acute myelogenous leukemia. One result of this translocation is a fusion between the AML1, MDS1, and EVI1 genes, which encodes a transcription factor of approximately 200 kDa. The role of the AML1/MDS1/EVI1 (AME) fusion gene in leukemogenesis is largely unknown. In this study, we analyzed the effect of the AME fusion gene in vivo by expressing it in mouse bone marrow cells via retroviral transduction. We found that mice transplanted with AME-transduced bone marrow cells suffered from an acute myelogenous leukemia (AML) 5–13 mo after transplantation. The disease could be readily transferred into secondary recipients with a much shorter latency. Morphological analysis of peripheral blood and bone marrow smears demonstrated the presence of myeloid blast cells and differentiated but immature cells of both myelocytic and monocytic lineages. Cytochemical and flow cytometric analysis confirmed that these mice had a disease similar to the human acute myelomonocytic leukemia. This murine model for AME-induced AML will help dissect the molecular mechanism of AML and the molecular biology of the AML1, MDS1, and EVI1 genes.