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We here investigated the effects of overexpressed superoxide dismutase (SOD)1 and amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants G93A and G147S in Neuro 2A (N2A) cell lines, and found a three-fold increase in lamellipodia either in cells cultured under differentiated or undifferentiated growth conditions. In undifferentiated N2A cells, SOD1 constructs promoted lamellipodial protrusions to similar extent as the overexpression of Rac1, and SOD1-mediated lamellipodia were prevented by coexpression of the N17 dominant-negative form of Rac1, or shRNA for a downstream effector of Rac1, the insulin receptor tyrosine kinase substrate p53 (IRSp53) or its binding partner LIN7. Moreover, no additive effect was measured by coexpression of the SOD1 constructs with Rac1, IRSp53 or LIN7. Collectively these data support a role for SOD1 in the regulation of Rac1-mediated lamellipodia pathway, a property fully retained by the two SOD1 mutants.

The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ∼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.

The α3β4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 β4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the β4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 β4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of β4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the β4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3β4 nicotinic receptor is more than an accessory subunit.

The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.

Studies with heterologous expression systems have shown that the α4β2 nicotinic acetylcholine receptor (nAChR) subtype can exist in two stoichiometries (with two [(α4)2(β2)3] or three [(α4)3(β2)2] copies of the α subunit in the receptor pentamer) which have different pharmacological and functional properties and are differently regulated by chronic nicotine treatment. However, the effects of nicotine treatment in vivo on native α4β2 nAChR stoichiometry are not well known. We investigated in C57BL/6 mice the in vivo effect of 14-day chronic nicotine treatment and subsequent withdrawal, on the subunit expression and β2/α4 subunit ratio of (3)H-epibatidine labeled α4β2*-nAChR in total homogenates of cortex and thalamus. We found that in basal conditions the ratio of the β2/α4 subunit in the cortex and thalamus is different indicating a higher proportion in receptors with (α4)2(β2)3 subunit stoichiometry in the thalamus. For cortex exposure to chronic nicotine elicited an increase in receptor density measured by (3)H-epibatidine binding, an increase in the α4 and β2 protein levels, and an increase in β2/α4 subunit ratio, that indicates an increased proportion of receptors with the (α4)2(β2)3 stoichiometry. For thalamus we did not find a significant increase in receptor density, α4 and β2 protein levels, or changes in β2/α4 subunit ratio. All the changes elicited by chronic nicotine in cortex were transient and returned to basal levels with an average half-life of 2.8 days following nicotine withdrawal. These data suggest that chronic nicotine exposure in vivo favors increased assembly of α4β2 nAChR containing three β2 subunits. A greater change in stoichiometry was observed for cortex (which has relatively low basal expression of (α4)2(β2)3 nAChR) than in thalamus (which has a relatively high basal expression of (α4)2(β2)3 nAChR).

Lung cancer is the leading cause of cancer-related deaths worldwide. Smoking accounts for approximately 70% of the cases of non- small cell lung cancer (NSCLC) and 90% of the cases of small-cell lung cancer (SCLC), although some patients develop lung cancer without a history of smoking. Nicotine is the most active addictive component of tobacco smoke. It does not initiate tumorigenesis in humans and rodents, but it alters the pathophysiology of lung cells by inducing the secretion of growth factors, neurotransmitters and cytokines, and promotes tumour growth and metastases by inducing cell cycle progression, migration, invasion, angiogenesis and the evasion of apoptosis. Most of these effects are a result of nicotine binding and activation of cell-surface neuronal nicotinic acetylcholine receptors (nAChRs) and downstream intracellular signalling cascades, and many are blocked by nAChR subtype-selective antagonists. Recent genome-wide association studies have revealed single nucleotide polymorphisms of nAChR subunits that influence nicotine dependence and lung cancer. This review describes the molecular basis of nAChR structural and functional diversity in normal and cancer lung cells, and the genetic alterations facilitating smoking-induced lung cancers. It also summarises current knowledge concerning the intracellular pathways activated by nicotine and other compounds present in tobacco smoke.

The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ∼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.

By means of immunofluorescence and subcellular fractionation experiments, we here demonstrate mitochondrial distribution of LIN7 and IRSp53 in HeLa cells. These peripheral proteins displayed a tight association with mitochondria and coimmunoprecipitated from mitochondrial fractions. In line with a role for LIN7 in the regulation of IRSp53 activity on actin dynamics, the morphology of mitochondria was similarly altered by changing the expression levels of either each protein or both, whereas mitochondrial morphology was preserved in cells overexpressing IRSp53 deleted of its binding domains for LIN7 (IRSp53Δ5) or for actin polymerisation modulators (IRSp53ΔSH3). In particular, the overexpression of full length LIN7 and/or IRSp53 increased the percentage of cells with short mitochondria, while downregulation of the endogenous proteins by shRNAs increased the amount of cells with elongated and perinuclear clustered mitochondria. These mitochondria were only partially resistant to fragmentation induced by dissipation of the mitochondrial membrane potential (i.e. treatment with sodium azide), whereas mitochondria were fully protected by the fission defective mutant Drp1 K38A. Overexpression of LIN7 or IRSp53 did not prevent the formation of hyperfused mitochondria in cells coexpressing the Drp1 K38A mutant, thus suggesting that LIN7-IRSp53 complex requires functional Drp1 to regulate mitochondrial morphology.

By means of morphological and biochemical criteria, we here provide evidence for the localization and function of premature ovarian failure, 1B (POF1B) in desmosomes. In monolayers of Caco-2 intestinal cells and in stratified HaCaT keratinocytes, endogenous POF1B colocalized with desmoplakin at desmosome plaques and in cytoplasmic particles aligned along intermediate filaments (IFs). POF1B predominantly co-fractionated with desmosomes and IF components and exhibited properties characteristic of desmosomes (i.e., detergent insolubility and calcium independence). The role of NH2 and COOH domains in the association of POF1B with desmosomes and IFs was revealed by transient expression of the truncated protein in Caco-2 cells and in cells lacking desmosomes. The function of POF1B in desmosomes was investigated in HaCaT keratinocytes stably downregulated for POF1B expression. Transmission electron microscopy analysis revealed a decrease in desmosome number and size, and desmosomes of the downregulated keratinocytes displayed weak electron-dense plaques. Desmosome alterations were associated with defects in cell adhesion, as revealed by the reduced resistance to mechanical stress in the dispase fragmentation assay. Moreover, desmosome localization of POF1B was restricted to granular layers in human healthy epidermis, whereas it largely increased in hyperproliferative human skin diseases, thus demonstrating the localization of POF1B also in desmosomes of multistratified epithelia.Journal of Investigative Dermatology advance online publication, 18 September 2014; doi:10.1038/jid.2014.327

Filopodia are dynamic, actin-rich finger-like structures that protrude from the cell membrane and play important roles in cell migration and neurite initiation and outgrowth. The insulin receptor substrate protein of 53 kDa (IRSp53) and the mammalian Diaphanous members of the formin family of proteins (mDia) are two key players in the formation of filopodia and neurites. IRSp53 is an adaptor protein that acts at the membrane:actin interface, coupling membrane deformation with F-actin polymerization. mDia formin proteins, instead, can nucleate and polymerize linear actin filaments. Emerging genetic and biochemical evidence indicate that there are multiple and independent pathways leading to filopodium and neurite formation, but the precise molecular components of these pathways remain ill-defined. We recently identified the PDZ domain-containing protein LIN7 as a novel regulator of IRSp53. We further showed that the association between these two proteins is required to promote the formation of filopodia and neurites independently from mDia formin proteins, highlighting novel mechanisms of filopodia and neurite formation.

The insulin receptor substrate protein of 53 kDa (IRSp53) is crucially involved in the formation of filopodia and neurites through mechanisms that have only partially been clarified. We have investigated the role of the small scaffold protein LIN7, which interacts with IRSp53. We found that formation of actin-filled protrusions in neuronal NSC34 cells and neurites in neuroblastoma N2A cells depends on motifs mediating the LIN7:IRSp53 association, as both the coexpression of LIN7 with IRSp53 or the expression of the L27-IRSp53 chimera (a fusion protein between IRSp53 and the LIN7L27 domain for plasma membrane protein complexes association) prevented actin-deficient protrusions induced by overexpressed IRSp53, and enhanced the formation of actin-filled protrusions. The regulatory role of LIN7 in IRSp53-mediated extension of filopodia in neuronal N2A cells was demonstrated by live-cell imaging experiments. Moreover, LIN7 silencing prevented the extension of filopodia and neurites, induced by ectopic expression of IRSp53 or serum starvation, respectively, in undifferentiated and differentiated N2A cells. The expression of full-length IRSp53 or the LIN7ΔPDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7-silenced cells. Conversely, defective neuritogenesis could be rescued by the expression of RNAi-resistant full-length LIN7 or chimeric L27-IRSp53. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100-insoluble complexes, otherwise occurring in differentiated cells. Collectively these data indicate that LIN7 is a novel regulator of IRSp53, and that the association of these proteins is required to promote the formation of actin-dependent filopodia and neurites.

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton. © 2011. Published by The Company of Biologists Ltd.