[Microscopic imaging of calcium ions with genetically encoded calcium indicators]. / Mikroskopowe obrazowanie jonów wapnia za pomoca genetycznie kodowanych sond.

Calcium is a second messenger that plays a key role in various cellular processes. Monitoring calcium levels is a prerequisite to their understanding. The first calcium indicators for microscopy were the luminescent protein aequorin and chemical probes. These indicators, however, have serious drawbacks, limiting their use in many types of experiments. Cloning of cDNA for the A. victoria GFP and creation of its first spectral variants has initiated the development of fluorescent, genetically encoded calcium indicators (GECI). They are composed of a fluorescent protein and a calcium-binding protein, usually calmodulin. The binding of calcium to the sensory domain of the indicator affects the fluorescent properties of the chromophore, which enables recording of calcium signals as fluorescent light. GECIs have many advantages and are essential for conducting long-term experiments in vivo. This article gives an overview of the currently available GECIs, the history of their development, applications and microscopic imaging systems.

Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

Familial Alzheimer's disease (FAD)-causing mutations in presenilins were shown to alter intracellular calcium dynamics, including store-operated calcium entry (SOCE). However, the involvement of FAD-linked amyloid precursor protein (APP) in SOCE remains controversial. Here, we used gain-of-function and loss-of-function approaches to shed light on this issue. We found that Jurkat cells, which exhibit prominent SOCE mediated by Orai channels, maintain low APP levels. The ectopic expression of APP, either with wildtype sequence or FAD-causing Swedish mutation, had no effect on SOCE induced by calcium store depletion with cyclopiazonic acid (CPA). The overproduction of C99 fragments, mimicking amyloidogenic processing of APP, also had no effect. Moreover, there was no alteration in the CPA-evoked SOCE upon APP knockdown in HeLa cells, which natively express 100-fold more APP than Jurkat cells. Consistently, we found no evidence for APP-dependent changes in the mRNA or protein levels of main SOCE components. Altogether, these results suggest that APP does not modulate Orai-dependent SOCE following quantitative calcium store depletion.

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum.

Ca(2+) is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca(2+) influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca(2+)-dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca(2+) concentration in the endoplasmic reticulum (ER). ER-resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca(2+) ATPase by increasing the ER Ca(2+) permeability. This results in diminished cytosolic and mitochondrial Ca(2+) signals upon stimulation of inositol 1,4,5-trisphosphate receptors and reduces Ca(2+) release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca(2+) release and augments sensitivity to apoptosis. Our findings indicate an important function of ER-resident TRPP2 in the modulation of intracellular Ca(2+) signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.

PRKCSH/80K-H, the protein mutated in polycystic liver disease, protects polycystin-2/TRPP2 against HERP-mediated degradation.

Autosomal dominant polycystic liver disease (PCLD) is caused by mutations of either PRKCSH or Sec63, two proteins associated with the endoplasmic reticulum (ER). Both proteins are involved in carbohydrate processing, folding and translocation of newly synthesized glycoproteins. It is postulated that defective quality control of proteins initiates endoplasmic reticulum-associated degradation (ERAD), which disrupts hepatic homeostasis in patients with PRKCSH or Sec63 mutations. However, the precise molecular mechanisms are not known. Here, we show that over-expression or depletion of PRKCSH in zebrafish embryos leads to pronephric cysts, abnormal body curvature and situs inversus. Identical phenotypic changes are induced by depletion or over-expression of TRPP2. Increased PRKCSH levels ameliorate developmental abnormalities caused by over-expressed TRPP2, whereas excess TRPP2 can compensate the loss PRKCSH, indicating that the proteins share a common signaling pathway. PRKCSH binds the C-terminal domain of TRPP2, and both proteins co-localize within the ER. Furthermore, PRKCSH interacts with Herp, and inhibits Herp-mediated ubiquitination of TRPP2. Our findings suggest that PRKCSH functions as a chaperone-like molecule, which prevents ERAD of TRPP2. Dysequilibrium between TRPP2 and PRKCSH may lead to cyst formation in PCLD patients with PRKCSH mutations, and thereby account for the overlapping manifestations observed in PCLD and autosomal dominant polycystic kidney disease.

Regulation of ciliary polarity by the APC/C.

Planar cell polarity signaling controls a variety of polarized cell behaviors. In multiciliated Xenopus epidermal cells, recruitment of Dishevelled (Dvl) to the basal body and its localization to the center of the ciliary rootlet are required to correctly position the motile cilia. We now report that the anaphase-promoting complex (APC/C) recognizes a D-box motif of Dvl and ubiquitylates Dvl on a highly conserved lysine residue. Inhibition of APC/C function by knockdown of the ANAPC2 subunit disrupts the polarity of motile cilia and alters the directionality of the fluid movement along the epidermis of the Xenopus embryo. Our results suggest that the APC/C activity enables cilia to correctly polarize in Xenopus epidermal cells.

TENT5 cytoplasmic noncanonical poly(A) polymerases regulate the innate immune response in animals.

Innate immunity is the first line of host defense against pathogens. Here, through global transcriptome and proteome analyses, we uncover that newly described cytoplasmic poly(A) polymerase TENT-5 (terminal nucleotidyltransferase 5) enhances the expression of secreted innate immunity effector proteins in Caenorhabditis elegans. Direct RNA sequencing revealed that multiple mRNAs with signal peptide-encoding sequences have shorter poly(A) tails in tent-5-deficient worms. Those mRNAs are translated at the endoplasmic reticulum where a fraction of TENT-5 is present, implying that they represent its direct substrates. Loss of tent-5 makes worms more susceptible to bacterial infection. Notably, the role of TENT-5 in innate immunity is evolutionarily conserved. Its orthologs, TENT5A and TENT5C, are expressed in macrophages and induced during their activation. Analysis of macrophages devoid of TENT5A/C revealed their role in the regulation of secreted proteins involved in defense response. In summary, our study reveals cytoplasmic polyadenylation to be a previously unknown component of the posttranscriptional regulation of innate immunity in animals.

Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel.

Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia.

Neuronal calcium signaling via store-operated channels in health and disease.

Store-operated calcium entry (SOCE) is the flow of calcium ions (Ca2+) into cells in response to the depletion of intracellular Ca2+ stores that reside predominantly in the endoplasmic reticulum (ER). The role of SOCE has been relatively well understood for non-excitable cells. It is mediated mostly by the ER Ca2+ sensor STIM1 and plasma membrane Ca2+ channel Orai1 and serves to sustain Ca2+ signaling and refill ER Ca2+ stores. In contrast, because of the complexity of Ca2+ influx mechanisms that are present in excitable cells, our knowledge about the function of neuronal SOCE (nSOCE) is still nascent. This review summarizes the available data on the molecular components of nSOCE and their relevance to neuronal signaling. We also present evidence of disturbances of nSOCE in neurodegenerative diseases (namely Alzheimer's disease, Huntington's disease, and Parkinson's disease) and traumatic brain injury. The emerging important role of nSOCE in neuronal physiology and pathology makes it a possible clinical target.

Knockdown of amyloid precursor protein increases calcium levels in the endoplasmic reticulum.

Familial Alzheimer's disease (AD) is caused by mutations in the genes that encode amyloid precursor protein (APP) and presenilins. Disturbances in calcium homeostasis have been observed in various cellular and animal models of AD and are proposed to underlie the pathogenesis of the disease. Furthermore, wildtype presenilins were shown to regulate endoplasmic reticulum (ER) calcium homeostasis, although their precise mechanism of action remains controversial. To investigate whether APP also affects ER calcium levels, we used RNA interference to target the APP gene in cultured T84 cells in combination with two types of ER calcium sensors. Using a genetically encoded calcium indicator, GEM-CEPIA1er, we found that APP-deficient cells exhibited elevated resting calcium levels in the ER and prolonged emptying of ER calcium stores upon the cyclopiazonic acid-induced inhibition of sarco-endoplasmic reticulum calcium-ATPase. These effects could be ascribed to lower ER calcium leakage rates. Consistent with these results, translocation of the endogenous ER calcium sensor STIM1 to its target channel Orai1 was delayed following ER calcium store depletion. Our data suggest a physiological function of APP in the regulation of ER calcium levels.

Molecular anatomy of the thalamic complex and the underlying transcription factors.

Thalamocortical loops have been implicated in the control of higher-order cognitive functions, but advances in our understanding of the molecular underpinnings of neocortical organization have not been accompanied by similar analyses in the thalamus. Using expression-based correlation maps and the manual mapping of mouse and human datasets available in the Allen Brain Atlas, we identified a few individual regions and several sets of molecularly related nuclei that partially overlap with the classic grouping that is based on topographical localization and thalamocortical connections. These new molecular divisions of the adult thalamic complex are defined by the combinatorial expression of Tcf7l2, Lef1, Gbx2, Prox1, Pou4f1, Esrrg, and Six3 transcription factor genes. Further in silico and experimental analyses provided the evidence that TCF7L2 might be a pan-thalamic specifier. These results provide substantial insights into the "molecular logic" that underlies organization of the thalamic complex.

Tyrosine phosphorylation modulates the activity of TRPV4 in response to defined stimuli.

Src family tyrosine kinases (SFKs) regulate the function of several transient receptor potential (TRP) family members, yet their role in the regulation of the vanilloid subfamily member 4 protein (TRPV4) remains controversial. TRPV4 is a calcium-permeable channel activated by numerous physical and chemical stimuli. Here we show that SFKs mediate tyrosine phosphorylation of TRPV4 in different cell lines. Using mass spectrometric analysis, we identified two novel phosphorylation sites in the cytosolic N- and C-terminal tails of TRPV4. Substitution of either tyrosine with phenylalanine led to a substantial reduction in the overall tyrosine phosphorylation level of TRPV4, suggesting that these two tyrosines constitute major phosphorylation sites. Both mutants efficiently localized to the plasma membrane, indicating that neither tyrosine is required for trafficking of TRPV4 in the secretory pathway. Analysis of the channel function demonstrated a crucial role of the N-terminal tyrosine residue in the activation of TRPV4 by heat, mechanical (shear) stress, hypotonic cell swelling, and phorbol 12-myristate 13-acetate, but not in the activation by synthetic ligand 4alpha-phorbol 12,13-didecanoate. Furthermore, the response of TRPV4 to phorbol 12-myristate 13-acetate was SFK-dependent. Because the SFK-mediated phosphorylation of the N-terminal tyrosine occurred before TRPV4 activation, tyrosine phosphorylation appears to sensitize rather than activate this channel. Reactive oxygen species, known to mediate inflammatory pain, strongly up-regulated TRPV4 phosphorylation in the presence of SFKs. Our findings indicate that tyrosine phosphorylation of TRPV4 represents an important modulatory mechanism, which may underlie the recently described function of TRPV4 in inflammatory hyperalgesia.

TRPP2 and TRPV4 form a polymodal sensory channel complex.

The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing constitutes the fundamental mechanism of cystogenesis.

OS-9 regulates the transit and polyubiquitination of TRPV4 in the endoplasmic reticulum.

Transient receptor potential (TRP) proteins constitute a family of cation-permeable channels that are formed by homo- or heteromeric assembly of four subunits. Despite recent progress in the identification of protein domains required for the formation of tetramers, the mechanisms governing TRP channel assembly, and biogenesis in general, remain largely elusive. In particular, little is known about the involvement of regulatory proteins in these processes. Here we report that OS-9, a ubiquitously expressed endoplasmic reticulum (ER)-associated protein, interacts with the cytosolic N-terminal tail of TRPV4. Using a combination of co-expression and knockdown approaches we have found that OS-9 impedes the release of TRPV4 from the ER and reduces its amount at the plasma membrane. Consistent with these in vitro findings, OS-9 protected zebrafish embryos against the detrimental effects of TRPV4 expression in vivo. A detailed analysis of the underlying mechanisms revealed that OS-9 preferably binds TRPV4 monomers and other ER-localized, immature variants of TRPV4 and attenuates their polyubiquitination. Thus, OS-9 regulates the secretory transport of TRPV4 and appears to protect TRPV4 subunits from the precocious ubiquitination and ER-associated degradation. Our data suggest that OS-9 functions as an auxiliary protein for TRPV4 maturation.

The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels.

TRPV4 is a widely expressed member of the transient receptor potential (TRP) family that facilitates Ca(2+) entry into nonexcitable cells. TRPV4 is activated by several stimuli, but it is largely unknown how the activity of this channel is terminated. Here, we show that ubiquitination represents an important mechanism to control the presence of TRPV4 at the plasma membrane. Ubiquitination of TRPV4 is dramatically increased by the HECT (homologous to E6-AP carboxyl terminus)-family ubiquitin ligase AIP4 without inducing degradation of this channel. Instead, AIP4 promotes the endocytosis of TRPV4 and decreases its amount at the plasma membrane. Consequently, the basal activity of TRPV4 is reduced despite an overall increase in TRPV4 levels. This mode of regulation is not limited to TRPV4. TRPC4, another member of the TRP channel family, is also strongly ubiquitinated in the presence of AIP4, leading to the increased intracellular localization of TRPC4 and the reduction of its basal activity. However, ubiquitination of several other TRP channels is not affected by AIP4, demonstrating that AIP4-mediated regulation is a unique property of select TRP channels.