Defective Entry Research Articles

Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein.

The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.

Gap junctions mediate discrete regulatory steps during fly spermatogenesis.

Gametogenesis requires coordinated signaling between germ cells and somatic cells. We previously showed that Gap junction (GJ)-mediated soma-germline communication is essential for fly spermatogenesis. Specifically, the GJ protein Innexin4/Zero population growth (Zpg) is necessary for somatic and germline stem cell maintenance and differentiation. It remains unknown how GJ-mediated signals regulate spermatogenesis or whether the function of these signals is restricted to the earliest stages of spermatogenesis. Here we carried out comprehensive structure/function analysis of Zpg using insights obtained from the protein structure of innexins to design mutations aimed at selectively perturbing different regulatory regions as well as the channel pore of Zpg. We identify the roles of various regulatory sites in Zpg in the assembly and maintenance of GJs at the plasma membrane. Moreover, mutations designed to selectively disrupt, based on size and charge, the passage of cargos through the Zpg channel pore, blocked different stages of spermatogenesis. Mutations were identified that progressed through early germline and soma development, but exhibited defects in entry to meiosis or sperm individualisation, resulting in reduced fertility or sterility. Our work shows that specific signals that pass through GJs regulate the transition between different stages of gametogenesis.

WDR5-HOTTIP Histone Modifying Complex Regulates Neural Migration and Dendrite Polarity of Pyramidal Neurons via Reelin Signaling.

WD-repeat domain 5 (WDR5), a core component of histone methyltransferase complexes, is associated with Kabuki syndrome and Kleefstra syndrome that feature intellectual disability and neurodevelopmental delay. Despite its critical status in gene regulation and neurological disorders, the role of WDR5 in neural development is unknown. Here we show that WDR5 is required for normal neuronal placement and dendrite polarization in the developing cerebral cortex. WDR5 knockdown led to defects in both entry into the bipolar transition of pyramidal neurons within the intermediate zone and radial migration into cortical layers. Moreover, WDR5 deficiency disrupted apical and basal polarity of cortical dendrites. Aberrant dendritic spines and synapses accompanied the dendrite polarity phenotype. WDR5 deficiency reduced expression of reelin signaling receptors, ApoER and VdldR, which were associated with abnormal H3K4 methylation and H4 acetylation on their promoter regions. Finally, an lncRNA, HOTTIP, was found to be a partner of WDR5 to regulate dendritic polarity and reelin signaling via histone modification. Our results demonstrate a novel role for WDR5 in neuronal development and provide mechanistic insights into the neuropathology associated with histone methyltransferase dysfunction.

Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission

Malaria‐causing parasites rely on an actin–myosin‐based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N‐terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent‐infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.

Whi7/Srl3 polymorphisms reveal its role in cell size and quiescence.

Whi5 and Srl3/Whi7 are related proteins that resulted from the whole genome duplication of S. cerevisiae (Wolfe and Shields 1997). Whi5 plays an Rb-like function in binding and inhibiting the late G1 transcription that promotes progression from G1 to S (Costanzo et al. 2004; de Bruin et al. 2004). Whi7 can also associate with G1 transcription complexes and promotes G1 arrest when overproduced (Gomar-Alba et al. 2017), but its transcription is primarily induced by stress (Ragni et al. 2011; Mendez et al. 2020). We have used polymorphisms in two laboratory yeast strains to uncover novel functions of Whi7 in log and quiescent cells. These include small cell size during log phase and defects in entry, maintenance and recovery from quiescence.

Tetraspanin CD81 regulates HSV-1 infection.

Different members of the tetraspanin superfamily have been described to regulate different virus infectious cycles at several stages: viral entry, viral replication or virion exit or infectivity. In addition, tetraspanin CD81 regulates HIV reverse transcription through its association with the dNTP hydrolase SAMHD1. Here we aimed at analysing the role of CD81 in Herpes simplex virus 1 infectivity using a neuroblastoma cell model. For this purpose, we generated a CD81 KO cell line using the CRISPR/Cas9 technology. Despite being CD81 a plasma membrane protein, CD81 KO cells showed no defects in viral entry nor in the expression of early protein markers. In contrast, glycoprotein B and C, which require viral DNA replication for their expression, were significantly reduced in CD81 KO infected cells. Indeed, HSV-1 DNA replication and the formation of new infectious particles were severely compromised in CD81 KO cells. We could not detect significant changes in SAMHD1 total expression levels, but a relocalization into endosomal structures was observed in CD81 KO cells. In summary, CD81 KO cells showed impaired viral DNA replication and produced greatly diminished viral titers.

The fission yeast Greatwall-Endosulfine pathway is required for proper quiescence/G0 phase entry and maintenance.

Cell proliferation and cellular quiescence/G0 phase must be regulated in response to intra-/extracellular environments, and such regulation is achieved by the orchestration of protein kinases and protein phosphatases. Here, we investigated fission yeast potential orthologs (Cek1, Ppk18 and Ppk31) of the metazoan Greatwall kinase (Gwl), which inhibits type-2A protein phosphatase with B55 subunit (PP2AB55 ) by phosphorylating and activating the PP2AB55 inhibitors, α-endosulfine/ARPP-19 (Ensa/ARPP-19). Gwl and Ensa/ARPP-19 regulate mitosis; however, we found Ppk18, Cek1 and Mug134/Igo1, the counterpart of Ensa/ARPP-19, are not essential for normal mitosis but regulate nitrogen starvation (-N)-induced proper G0 entry and maintenance. Genetic and biochemical analyses indicated that the conserved Gwl site (serine 64) was phosphorylated in the G0 phase in a Ppk18-dependent manner, and the phosphorylated Mug134/Igo1 inhibited PP2AB55 in vitro. The alanine substitution of the serine 64 caused defects in G0 entry and maintenance as well as the mug134/igo1+ deletion. These results indicate that PP2AB55 activity must be regulated properly to establish the G0 phase. Consistently, simultaneous deletion of the B55 gene with mug134/igo1+ partially rescued the Mug134/Igo1 mutant phenotype. We suggest that in fission yeast, PP2AB55 regulation by the Ppk18-Mug134/Igo1 pathway is required for G0 entry and establishment of robust viability during the G0 phase.

PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells.

The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)-containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY-containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock-down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re-distribution of HuR, which in turn led to increased cap-independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2-HuR-p27 pathway.

Global Promoter Targeting of a Conserved Lysine Deacetylase for Transcriptional Shutoff during Quiescence Entry

Quiescence is a conserved cell-cycle state characterized by cell-cycle arrest, increased stress resistance, enhanced longevity, and decreased transcriptional, translational, and metabolic output. Although quiescence plays essential roles in cell survival and normal differentiation, the molecular mechanisms leading to this state are not well understood. Here, we determined changes in the transcriptome and chromatin structure of S. cerevisiae upon quiescence entry. Our analyses revealed transcriptional shutoff that is far more robust than previously believed and an unprecedented global chromatin transition, which are tightly correlated. These changes require Rpd3 lysine deacetylase targeting to at least half of gene promoters via quiescence-specific transcription factors including Xbp1 and Stb3. Deletion of RPD3 prevents cells from establishing transcriptional quiescence, leading to defects in quiescence entry and shortening of chronological lifespan. Our results define a molecular mechanism for global reprogramming of transcriptome and chromatin structure for quiescence driven by a highly conserved chromatin regulator.

Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection

Several enveloped viruses exploit host pathways, such as the cellular endosomal sorting complex required for transport (ESCRT) machinery, for their assembly and release. The influenza A virus (IAV) matrix protein binds to the ESCRT-I complex, although the involvement of early ESCRT proteins such as Tsg101 in IAV trafficking remain to be established. We find that Tsg101 can facilitate IAV trafficking, but this is effectively restricted by the interferon (IFN)-stimulated protein ISG15. Cytosol from type I IFN-treated cells abolished IAV hemagglutinin (HA) transport to the cell surface in infected semi-intact cells. This inhibition required Tsg101 and could be relieved with deISGylases. Tsg101 is itself ISGylated in IFN-treated cells. Upon infection, intact Tsg101-deficient cells obtained by CRISPR-Cas9 genome editing were defective in the surface display of HA and for infectious virion release. These data support the IFN-induced generation of a Tsg101- and ISG15-dependent checkpoint in the secretory pathway that compromises influenza virus release.

Human Cytomegalovirus Infection of Langerhans-Type Dendritic Cells Does Not Require the Presence of the gH/gL/UL128-131A Complex and Is Blocked after Nuclear Deposition of Viral Genomes in Immature Cells

Human cytomegalovirus (CMV) enters its host via the oral and genital mucosae. Langerhans-type dendritic cells (LC) are the most abundant innate immune cells at these sites, where they constitute a first line of defense against a variety of pathogens. We previously showed that immature LC (iLC) are remarkably resistant to CMV infection, while mature LC (mLC) are more permissive, particularly when exposed to clinical-strain-like strains of CMV, which display a pentameric complex consisting of the viral glycoproteins gH, gL, UL128, UL130, and UL131A on their envelope. This complex was recently shown to be required for the infection of immature monocyte-derived dendritic cells. We thus sought to establish if the presence of this complex is also necessary for virion penetration of LC and if defects in entry might be the source of iLC resistance to CMV. Here we report that the efficiency of LC infection is reduced, but not completely abolished, in the absence of the pentameric complex. While virion penetration and nuclear deposition of viral genomes are not impaired in iLC, the transcription of the viral immediate early genes UL122 and UL123 and of the delayed early gene UL50 is substantially lower than that in mLC. Together, these data show that the UL128, UL130, and UL131A proteins are dispensable for CMV entry into LC and that progression of the viral cycle in iLC is restricted at the step of viral gene expression.

Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)(1-23), human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA(1-32), required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.

Identification of Genes from the Fungal Pathogen Cryptococcus neoformans Related to Transmigration into the Central Nervous System

BackgroundA mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis.Methodology/Principal FindingsTwo cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the “Trojan horse” model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA.Conclusions/SignificanceThis research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry.

Cks1 Promotion of S Phase Entry and Proliferation Is Independent of p27Kip1 Suppression

Cks1 is an activator of the SCF(Skp2) ubiquitin ligase complex that targets the cell cycle inhibitor p27(Kip1) for degradation. The loss of Cks1 results in p27(Kip1) accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27(Kip1). Specifically, Cks1(-/-); p27(Kip1-/-) mouse embryonic fibroblasts retain defects in the G(1)-S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27(Kip1) that is manifest in various organs of p27(Kip1-/-) mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1(-/-) cells are p27(Kip1) dependent. Collectively, these findings establish p27(Kip1)-independent functions of Cks1 in regulating the G(1)-S transition.

Actin depolymerizing factor controls actin turnover and gliding motility inToxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

Yeast lsm pro-apoptotic mutants show defects in S-phase entry and progression

Expression of the histone genes is tightly coupled to rates of DNA synthesis in yeast and histone mRNAs are modulated both transcriptionally and post-transcriptionally. Trf4 and Trf5, poly(A) polymerases, that mediates polyadenylation and consequent degradation) and Rrp6, an exosome component, play a role in the regulation of histone mRNA levels. In this paper we show that in the mRNA degradation mutant Kllsm4D1, histone mRNAs are induced early in the S-phase and maintained at high level all along the entire cell cycle due to a delay in the exit from S-phase and/or entry into M-phase. The over-expression of the HIR1 gene (Histone transcriptional repressor), previously isolated as a multicopy suppressor of the apoptotic phenotypes observed in Kllsm4D1, can also restore the normal cycling of histone genes expression. We also found that low doses of hydroxyurea neutralize the onset of the apoptotic phenotypes in Kllsm4D1, as well in another mRNA decapping mutants (lsm1) and, in addition, increase the chronological lifespan in both strains suggesting that an entry delay into the S phase can recover some cellular defects in decapping mutants.

A Cellular Memory of Developmental History Generates Phenotypic Diversity in C. elegans

Early life experiences have a major impact on adult phenotypes [1-3]. However, the mechanisms by which animals retain a cellular memory of early experience are not well understood. Here we show that adult wild-type Caenorhabditis elegans that transiently pass through the stress-resistant dauer larval stage exhibit distinct gene expression profiles and life history traits, as compared to adult animals that bypassed this stage. Using chromatin immunoprecipitation experiments coupled with massively parallel sequencing, we found that genome-wide levels of specific histone tail modifications are markedly altered in postdauer animals. Mutations in subsets of genes implicated in chromatin remodeling abolish, or alter, the observed changes in gene expression and life history traits in postdauer animals. Modifications to the epigenome as a consequence of early experience may contribute in part to a memory of early experience and generate phenotypic variation in an isogenic population.

A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRDeltagO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRDeltagO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRDeltagO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRDeltagO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRDeltagO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.

EKLF/KLF1 Controls Cell Cycle Entry via Direct Regulation of E2f2

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

Lambert–Eaton myasthenic syndrome: Search for alternative autoimmune targets and possible compensatory mechanisms based on presynaptic calcium homeostasis

The Lambert–Eaton myasthenic syndrome (LEMS) is a disease of neuromuscular transmission in which autoantibodies against the P/Q-type voltage-gated calcium channel (VGCC) at the presynaptic nerve terminal play a major role in decreasing quantal release of acetylcholine (ACh), resulting in skeletal muscle weakness and autonomic symptoms. It is associated with cancer, particularly small-cell lung carcinoma (SCLC), in 50–60% of LEMS patients; the nerve terminal and carcinoma cells apparently share a common antigen (VGCC), suggesting an immunological cross-reactivity that may lead to the neurological abnormality. Non-tumor LEMS has a strong association with HLA-DR3-B8. In approximately 15% of LEMS patients, no anti-P/Q-type VGCC antibodies are found, suggesting recognition of other targets(s). The VGCC-associated protein synaptotagmin could be one candidate, because it acts as an exocytotic calcium receptor, is implicated in fast ACh release; its N-terminus is exposed extracellularly during exocytosis and it is expressed in SCLC. Antibodies against synaptotagmin-1 were detected in both anti-VGCC-positive and -negative LEMS patients (20%), and it can be immunogenic, allowing induction of an animal model of LEMS. Another candidate target is the M1-type presynaptic muscarinic ACh receptor (M1 mAChR), also expressed extracellularly on motor nerve terminals; it modulates cholinergic transmission, linking to P/Q-type VGCC. In our series of 25 LEMS patients with and without SCLC, anti-M1 mAChR antibodies were prevalent in both anti-VGCC-positive and -negative LEMS patients. Autonomic symptoms seemed more frequent in the latter; serum from one of them passively transferred LEMS-type electrophysiological defects to mice. As a compensatory mechanism, researchers in Oxford suggested a shift in the dependence of ACh release from the P/Q-type to other types of VGCC. We have also focused on G protein-coupled mAChRs and neurotrophins, which may affect both P/Q-type VGCC and clathrin-independent “kiss-and-run” synaptic vesicle recycling (fast-mode of endocytosis) via protein kinase C activation. We hypothesize that these signaling cascades help to compensate for the immune-mediated defects in calcium entry in LEMS, compensation that may frequently be restricted by the coincident anti-M1 mAChR antibodies in this disease.