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Molecular Biology of the Cell Jun 2023β-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β-spectrin...
β-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, β-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing β-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of β-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce β-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that β-spectrin acts upstream of these pathways as loss of β-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate β-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.
Topics: Animals; Mice; Endothelial Cells; Growth Differentiation Factor 2; Neovascularization, Physiologic; Signal Transduction; Spectrin
PubMed: 37126382
DOI: 10.1091/mbc.E23-02-0064 -
Antioxidants (Basel, Switzerland) Apr 2023Red blood cell (RBC) deformability is the ability of cells to modulate their shape to ensure transit through narrow capillaries of the microcirculation. A loss of...
Red blood cell (RBC) deformability is the ability of cells to modulate their shape to ensure transit through narrow capillaries of the microcirculation. A loss of deformability can occur in several pathological conditions, during natural RBC aging through an increase in membrane protein phosphorylation, and/or through the structural rearrangements of cytoskeletal proteins due to oxidative conditions, with a key role played by band 3. Due to the close relationship between aging and oxidative stress, flavonoid-rich foods are good candidates to counteract age-related alterations. This study aims to verify the beneficial role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human RBCs. To this end, band 3 phosphorylation and structural rearrangements in membrane cytoskeleton-associated proteins, namely spectrin, ankyrin, and/or protein 4.1, are analyzed in RBCs treated with 100 mM d-Gal for 24 h, with or without pre-incubation with 10 μg/mL Açaì extract for 1 h. Furthermore, RBC deformability is also measured. Tyrosine phosphorylation of band 3, membrane cytoskeleton-associated proteins, and RBC deformability (elongation index) are analyzed using western blotting analysis, FACScan flow cytometry, and ektacytometry, respectively. The present data show that: (i) Açaì berry extract restores the increase in band 3 tyrosine phosphorylation and Syk kinase levels after exposure to 100 mM d-Gal treatment; and (ii) Açaì berry extract partially restores alterations in the distribution of spectrin, ankyrin, and protein 4.1. Interestingly, the significant decrease in membrane RBC deformability associated with d-Gal treatment is alleviated by pre-treatment with Açaì extract. These findings further contribute to clarify mechanisms of natural aging in human RBCs, and propose flavonoid substances as potential natural antioxidants for the treatment and/or prevention of oxidative-stress-related disease risk.
PubMed: 37107223
DOI: 10.3390/antiox12040848 -
Frontiers in Neuroanatomy 2023The axon initial segment (AIS) is the site of action potential initiation and important for the integration of synaptic input. Length and localization of the AIS are...
The axon initial segment (AIS) is the site of action potential initiation and important for the integration of synaptic input. Length and localization of the AIS are dynamic, modulated by afferent activity and contribute to the homeostatic control of neuronal excitability. Synaptopodin is a plasticity-related protein expressed by the majority of telencephalic neurons. It is required for the formation of cisternal organelles within the AIS and an excellent marker to identify these enigmatic organelles at the light microscopic level. Here we applied 2 h of high frequency stimulation of the medial perforant path in rats to induce a strong long-term potentiation of dentate gyrus granule cells. Immunolabeling for βIV-spectrin and synaptopodin were performed to study structural changes of the AIS and its cisternal organelles. Three-dimensional analysis of the AIS revealed a shortening of the AIS and a corresponding reduction of the number of synaptopodin clusters. These data demonstrate a rapid structural plasticity of the AIS and its cisternal organelles to strong stimulation, indicating a homeostatic response of the entire AIS compartment.
PubMed: 37090138
DOI: 10.3389/fnana.2023.1125623 -
Nature Immunology Jun 2023Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T...
Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 β-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8 T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of βII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-βII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.
Topics: Animals; Mice; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Movement; Immunotherapy, Adoptive; Lymphocyte Function-Associated Antigen-1; Receptors, Chimeric Antigen; Spectrin; Humans; Female
PubMed: 37069398
DOI: 10.1038/s41590-023-01498-x -
Cell Apr 2023The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of...
The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/β-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems.
Topics: Animals; Humans; Actin Cytoskeleton; Actins; Cytoskeleton; Erythrocytes; Spectrin; Swine
PubMed: 37044097
DOI: 10.1016/j.cell.2023.03.017 -
BMC Cancer Apr 2023Nonerythrocytic spectrin beta 1 (SPTBN1) is an important cytoskeletal protein that involves in normal cell growth and development via regulating TGFβ/Smad signaling...
BACKGROUND
Nonerythrocytic spectrin beta 1 (SPTBN1) is an important cytoskeletal protein that involves in normal cell growth and development via regulating TGFβ/Smad signaling pathway, and is aberrantly expressed in various cancer types. But, the exact role of SPTBN1 in pan-cancer is still unclear. This report aimed to display expression patterns and prognostic landscapes of SPTBN1 in human cancers, and further assess its prognostic/therapeutic value and immunological role in kidney renal carcinoma (KIRC) and uveal melanoma (UVM).
METHODS
We firstly analyzed expression patterns and prognostic landscapes of SPTBN1 in human cancers using various databases and web-based tools. The relationships between SPTBN1 expression and survival/tumor immunity in KIRC and UVM were further investigated via R packages and TIMER 2.0 platform. The therapeutic roles of SPTBN1 in KIRC and UVM were also explored via R software. Following this, the prognostic value and cancer immunological role of SPTBN1 in KIRC and UVM were validated in our cancer patients and GEO database.
RESULTS
Overall, cancer tissue had a lower expression level of SPTBN1 frequently in pan-cancer, compared with those in adjacent nontumor one. SPTBN1 expression often showed a different effect on survival in pan-cancer; upregulation of SPTBN1 was protective to the survival of KIRC individuals, which was contrary from what was found in UVM patients. In KIRC, there were significant negative associations between SPTBN1 expression and pro-tumor immune cell infiltration, including Treg cell, Th2 cell, monocyte and M2-macrophage, and expression of immune modulator genes, such as tumor necrosis factor superfamily member 9 (TNFSF9); while, in UVM, these correlations exhibited opposite patterns. The following survival and expression correlation analysis in our cancer cohorts and GEO database confirmed these previous findings. Moreover, we also found that SPTBN1 was potentially involved in the resistance of immunotherapy in KIRC, and the enhance of anti-cancer targeted treatment in UVM.
CONCLUSIONS
The current study presented compelling evidence that SPTBN1 might be a novel prognostic and therapy-related biomarker in KIRC and UVM, shedding new light on anti-cancer strategy.
Topics: Humans; Carcinoma, Renal Cell; Spectrin; Clinical Relevance; Kidney Neoplasms; Kidney
PubMed: 37013511
DOI: 10.1186/s12885-023-10789-3 -
Cellular & Molecular Biology Letters Mar 2023Phosphatidylserine is translocated to the inner leaflet of the phospholipid bilayer membrane by the flippase function of type IV P-tape ATPase (P4-ATPase), which is...
BACKGROUND
Phosphatidylserine is translocated to the inner leaflet of the phospholipid bilayer membrane by the flippase function of type IV P-tape ATPase (P4-ATPase), which is critical to maintain cellular stability and homeostasis. Transmembrane protein 30A (TMEM30A) is the β-subunit of P4-ATPase. Loss of P4-ATPase function causes sensorineural hearing loss and visual dysfunction in human. However, the function of TMEM30A in the auditory system is unclear.
METHODS
P4-ATPase subtype expression in the cochlea was detected by immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) at different developmental stages. Hair cell specific TMEM30A knockout mice and wild-type littermates were used for the following functional and morphological analysis. Auditory function was evaluated by auditory brainstem response. We investigated hair cell and stereocilia morphological changes by immunofluorescence staining. Scanning electron microscopy was applied to observe the stereocilia ultrastructure. Differentially expressed transcriptomes were analyzed based on RNA-sequencing data from knockout and wild-type mouse cochleae. Differentially expressed genes were verified by qRT-PCR.
RESULTS
TMEM30A and subtypes of P4-ATPase are expressed in the mouse cochlea in a temporal-dependent pattern. Deletion of TMEM30A in hair cells impaired hearing onset due to progressive hair cell loss. The disrupted kinocilia placement and irregular distribution of spectrin-α in cuticular plate indicated the hair cell planar polarity disruption in TMEM30A deletion hair cells. Hair cell degeneration begins at P7 and finishes around P14. Transcriptional analysis indicates that the focal adhesion pathway and stereocilium tip-related genes changed dramatically. Without the TMEM30A chaperone, excessive ATP8A2 accumulated in the cytoplasm, leading to overwhelming endoplasmic reticulum stress, which eventually contributed to hair cell death.
CONCLUSIONS
Deletion of TMEM30A led to disrupted planar polarity and stereocilia bundles, and finally led to hair cell loss and auditory dysfunction. TMEM30A is essential for hair cell polarity maintenance and membrane homeostasis. Our study highlights a pivotal role of TMEM30A in the postnatal development of hair cells and reveals the possible mechanisms underlying P4-ATPase-related genetic hearing loss.
Topics: Mice; Animals; Humans; Cell Polarity; Mice, Knockout; Cochlea; Adenosine Triphosphatases; Membrane Proteins
PubMed: 36959542
DOI: 10.1186/s11658-023-00437-w -
Science Advances Mar 2023Immunoglobulin A (IgA) nephropathy (IgAN) is the most common type of primary glomerulonephritis, often progressing to renal failure. IgAN is triggered by IgA deposition...
Immunoglobulin A (IgA) nephropathy (IgAN) is the most common type of primary glomerulonephritis, often progressing to renal failure. IgAN is triggered by IgA deposition in the glomerular mesangium by an undefined mechanism. Here, we show that grouped ddY (gddY) mice, a spontaneous IgAN model, produce serum IgA against mesangial antigens, including βII-spectrin. Most patients with IgAN also have serum anti-βII-spectrin IgA. As in patients with IgAN, IgA plasmablasts accumulate in the kidneys of gddY mice. IgA antibodies cloned from the plasmablasts carry substantial V-region mutations and bind to βII-spectrin and the surface of mesangial cells. These IgAs recognize transfected and endogenous βII-spectrin exposed on the surface of embryonic kidney-derived cells. Last, we demonstrate that the cloned IgA can bind selectively to glomerular mesangial regions in situ. The identification of IgA autoantibody and its antigen in IgAN provides key insights into disease onset and redefines IgAN as a tissue-specific autoimmune disease.
Topics: Mice; Animals; Glomerulonephritis, IGA; Mesangial Cells; Spectrin; Immunoglobulin A; Autoantibodies
PubMed: 36947618
DOI: 10.1126/sciadv.add6734 -
Molecular and Cellular Neurosciences Jun 2023Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including...
Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including prevention of protein misfolding is essential for cellular activity and health. Molecular chaperones are key actors in proteostasis. BRICHOS domain is an intramolecular chaperone that also interferes with several aggregation-prone proteins including amyloid β (Aβ), involved in Alzheimer's disease (AD). To extend the knowledge about Bri2 BRICHOS interactome we here used recombinant human (rh) Bri2 BRICHOS-mCherry fusion protein to probe for potential binding partners. Firstly, exogenously added Bri2 BRICHOS-mCherry was used to stain brain sections of wildtype and amyloid precursor protein (App) knock-in AD mice exhibiting robust Aβ pathology. Unexpectedly, we found that rh Bri2 BRICHOS-mCherry stained the cytoplasm of neurons which are devoid of Aβ deposits. To identify these intraneuronal proteins that bind to the rh Bri2 BRICHOS domain, we performed co-immunoprecipitation (co-IP) of mouse brain hippocampi homogenates using the Bri2 BRICHOS-mCherry probe and analyzed co-IP proteins by LC-MS/MS. This identified several cytoskeletal proteins including spectrin alpha and beta chain, drebrin, tubulin β3, and β-actin as binding partners. The interactions were confirmed by a second round of pulldown experiments using rh Bri2 BRICHOS linked to magnetic beads. The interaction of rh Bri2 BRICHOS and tubulin β3 was further investigated by staining both mouse brain sections and SH-SY5Y neuroblastoma cells with rh Bri2 BRICHOS-mCherry and tubulin β3 immunostaining, which revealed partial co-localization. These data suggest a possible interplay of extracellular chaperone Bri2 BRICHOS domain in the intracellular space including the cytoskeleton.
Topics: Animals; Humans; Mice; Alzheimer Disease; Amyloid beta-Peptides; Chromatography, Liquid; Cytoskeletal Proteins; Membrane Glycoproteins; Molecular Chaperones; Neuroblastoma; Tandem Mass Spectrometry; Tubulin
PubMed: 36935047
DOI: 10.1016/j.mcn.2023.103843