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Transverse boundaries divide the mammalian cerebellar cortex into transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. This topography is highly conserved across the Mammalia. Bats have a remarkable cerebellum with presumed adaptations to flight and to echolocation, but nothing is known of its compartmentation. We have therefore used two Purkinje cell compartmentation antigens, zebrin II/aldolase C and phospholipase Cbeta4, to reveal the topography of the cerebellum in microchiropteran bats. Three species of bat were studied, Lasiurus cinereus, Lasionycteris noctivagans, and Eptesicus fuscus. A reproducible pattern of zones and stripes was revealed that is similar across the three species. The architecture of the bat cerebellum conforms to the ground plan of other mammals. However, two exceptions to the highly conserved mammalian architectural plan were revealed. First, many Purkinje cells in lobule I express zebrin II. A zebrin II-immunopositive lobule I has not been seen previously in mammals but is characteristic of the avian cerebellum. Second, lobules VI-VII comprise the large central zone. Within the central zone two subdomains are evident, a small anterior subdomain (lobule VI) in which Purkinje cells are predominantly zebrin II-immunopositive/PLCbeta4-immunonegative, as in other mammals, and a posterior subdomain (lobule VII), in which alternating zebrin II/phospholipase Cbeta4 stripes are prominent.
The plasma membrane of mammalian cells contains detergent-resistant membrane rafts enriched in glycosphingolipids and cholesterol. Although several important signaling molecules have been found in such rafts, evidence documenting a functional role for their localization has been scarce. Using a fractionation scheme that preserves tyrosine phosphorylation, we show that T cell activation leads to a striking compartmentation in the rafts of activated T cell receptor and associated signal-transducing molecules. Conditions that reversibly disrupt raft structure either by dispersing their contents or by forcing their internalization reversibly disrupt the earliest steps of T cell activation. Thus, raft integrity is a prerequisite for efficient T cell receptor signal transduction.
Despite the apparent uniformity in cellular composition of the mammalian cerebellar cortex, a complex topography is revealed by several expression patterns. Zebrin II, a polypeptide antigen identified as aldolase C, is one such marker which, in several species of mammals, is restricted to a subset of Purkinje cells that are clustered together to form a symmetrical and reproducible array of zones and stripes. In rodents the cerebellar cortex is divided into four transverse zones--anterior, central, posterior, and nodular. Each transverse zone is further subdivided mediolaterally into an array of parasagittal stripes. The similar zone and stripe organization partitions the hemispheres. Based upon a novel whole mount immunohistochemical staining procedure, we have now identified homologous zones and stripes in the feline cerebellum. In the cat cerebellum the somata of most Purkinje cells express zebrin II but parasagittal stripes may still be delineated owing to the alternating high and low zebrin II expression levels in the dendritic arbors. As in rodents, the cat cerebellum consists of four transverse zones with each zone subdivided into a unique combination of zebrin II parasagittal stripes, suggesting that a common architecture underlies the organization of the mammalian cerebellum.
Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.
The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.
The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens-zebrin II/aldolase C and phospholipase Cbeta4 (PLCbeta4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLCbeta4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-VIa) were distinguished. A central zone (CZ; approximately lobules VIa-VIIIa,b) and a posterior zone (PZ; approximately lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds.
Many diseases are linked to dysregulation of the striatum. Striatal function depends on neuronal compartmentation into striosomes and matrix. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3, using the Nr4a1-EGFP striosome reporter mouse. Focusing on the striosome, we validated the localization and/or role of Irx1, Foxf2, Olig2, and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of Olig2. These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.
Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.
Maintenance of a balanced expression of the two isoforms of the transcription factor GATA-1, the full-length protein (GATA-1FL ) and a shorter isoform (GATA-1 S ), contributes to control hematopoiesis, whereas their dysregulation can alter the differentiation/proliferation potential of hematopoietic precursors thereby eventually leading to a variety of hematopoietic disorders. Although it is well established that these isoforms play opposite roles in these remarkable processes, most of the molecular pathways involved remain unknown. Here, we demonstrate that GATA-1FL and GATA-1S are able to differently influence intracellular redox states and reactive oxygen species (ROS) compartmentation in the erythroleukemic K562 cell line, thus shedding novel mechanistic insights into the processes of cell proliferation and apoptosis resistance in myeloid precursors. Furthermore, given the role played by ROS signaling as a strategy to escape apoptosis and evade cell-mediated immunity in myeloid cells, this study highlights a mechanism through which aberrant expression of GATA-1 isoforms could play a role in the leukemogenic process.
The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.
In the metabolic syndrome, glucocorticoid activity is increased, but circulating levels show little change. Most of blood glucocorticoids are bound to corticosteroid-binding globulin (CBG), which liver expression and circulating levels are higher in females than in males. Since blood hormones are also bound to blood cells, and the size of this compartment is considerable for androgens and estrogens, we analyzed whether sex or eating a cafeteria diet altered the compartmentation of corticosterone in rat blood. The main corticosterone compartment in rat blood is that specifically bound to plasma proteins, with smaller compartments bound to blood cells or free. Cafeteria diet increased the expression of liver CBG gene, binding plasma capacity and the proportion of blood cell-bound corticosterone. There were marked sex differences in blood corticosterone compartmentation in rats, which were unrelated to testosterone. The use of a monoclonal antibody ELISA and a polyclonal Western blot for plasma CBG compared with both specific plasma binding of corticosterone and CBG gene expression suggested the existence of different forms of CBG, with varying affinities for corticosterone in males and females, since ELISA data showed higher plasma CBG for males, but binding and Western blot analyses (plus liver gene expression) and higher physiological effectiveness for females. Good cross-reactivity to the antigen for polyclonal CBG antibody suggests that in all cases we were measuring CBG. The different immunoreactivity and binding affinity may help explain the marked sex-related differences in plasma hormone binding as sex-linked different proportions of CBG forms.
Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA sequencing of maize mesophyll and bundle sheath strands over a 24-h time course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene coexpression analyses suggested members of the DNA binding with one finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators and peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.
Despite the apparent uniformity in cellular composition of the adult cerebellar cortex, a complex heterogeneous pattern can be revealed by using biochemical markers. One example is zebrin II/aldolase C, which is expressed by a subset of Purkinje cells that form a highly reproducible array of stripes. Zebrin II/aldolase C immunohistochemistry has been used in both section and whole mount preparations to analyze the architecture of the hamster cerebellar cortex. As in other species studied, zebrin II immunoreactivity in the hamster cerebellum is restricted to a subset of Purkinje cells and, more weakly, to astrocytes. Based on the distribution of these Purkinje cell subsets the hamster cerebellar vermis was found to consist of four transverse zones-the anterior zone, central zone, posterior zone and nodular zone. Zebrin II/aldolase C is expressed uniformly in the central and nodular zones, and as parasagittal stripes in the anterior and central zones. A similar alternation of homogeneous and striped expression domains is seen in the hemispheres. The topography of the hamster cerebellar cortex as revealed by zebrin II expression domains closely resembles that reported in other mammals. Thus, a cerebellar zone-and-stripe topography appears to be conserved across the Mammalia.
Granule cells (GCs) are the most numerous cell type in the cerebellum and indeed, in the brain: at least 99% of all cerebellar neurons are granule cells. In this review article, we first consider the formation of the upper rhombic lip, from which all granule cell precursors arise, and the way by which the upper rhombic lip generates the external granular layer, a secondary germinal epithelium that serves to amplify the upper rhombic lip precursors. Next, we review the mechanisms by which postmitotic granule cells are generated in the external granular layer and migrate radially to settle in the granular layer. In addition, we review the evidence that far from being a homogeneous population, granule cells come in multiple phenotypes with distinct topographical distributions and consider ways in which the heterogeneity of granule cells might arise during development.
Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.
Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.
B cell linker protein (BLNK) and Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76) are adaptor proteins required for B cell receptor (BCR) and T cell receptor function, respectively. Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells. This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking. Supporting this idea, the BCR function was restored when a membrane-associated SLP-76 chimera was enforcedly localized to GEMs. Moreover, we demonstrate that addition of both linker for activation of T cells (LAT) and Grb2-related adaptor downstream of Shc (Gads) to SLP-76 allow SLP-76 to be recruited into GEMs, whereby the BCR function is reconstituted. The Gads function was able to be replaced by overexpression of Grb2. In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.
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