Biology subjects

Citi, S.

Publications and source records attributed to Citi, S..

4 recordsLinked to original sources

bioRxiv2026

Phosphorylation of the rod-tail hinge region of cingulin regulates its interaction with nonmuscle myosin-2B

The tight junction (TJ) protein cingulin binds directly to nonmuscle myosin 2B (NM2B) through sequences in its C-terminal rod-tail region and recruits it to tight junctions (TJ) to control membrane cortex mechanics, epithelial morphogenesis and cingulin conformation. However, the minimal sequence required for cingulin-NM2B interaction and how this interaction is regulated is not known. Here we identify a 19-aminoacid sequence at the hinge between the cingulin rod and tail that is required for cingulin-NM2B interaction, and we investigate the role of phosphorylation of Ser residues within this region in regulating this interaction. Immunofluorescence microscopy localization of NM2B in cingulin-KO cells rescued with mutant cingulin constructs shows that phospho-mimetic but not dephospho-mimetic cingulin mutants inhibit NM2B recruitment to junctions and downstream regulation of cingulin conformation and TJ tortuosity, correlating with cingulin-NM2B interaction, as determined by GST pulldown analysis. In contrast, either phospo-or dephospho-mimetic mutants of Ser residues within the cingulin head domain do not affect either NM2B recruitment to TJ, or cingulin conformation and localization in cells, or TJ membrane tortuosity. Finally, Ser residues within the hinge display the consensus sequence for protein kinases CK1 and CK2, and, through in vitro phosphorylation, site mutation analysis and use of inhibitors, we identify a complex interplay between CGN phospho-sites, with a prominent negative role of Ser1162 phosphorylation in the regulation of cingulin-NM2B interaction. In summary, we show that cingulin-NM2B interaction is regulated by cingulin phosphorylation within the hinge and identify a potential role for CK1 and CK2 kinases in cingulin phosphorylation.

cell biology↗

bioRxiv2021

PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which similarly to the junctional protein PLEKHA7 bind to PDZD11 N-terminus through their WW domains. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable copper with the labile copper probe CF4 shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

cell biology↗

bioRxiv2020

Cingulin unfolds ZO-1 and organizes myosin-2B and gamma-actin to mechanoregulate apical and tight junction membranes

How junctional proteins regulate the mechanics of the plasma membrane and how actin and myosin isoforms are selectively localized at epithelial cell-cell junctions is poorly understood. Here we show by atomic force indentation microscopy, immunofluorescence analysis and FLIM membrane tension imaging that the tight junction (TJ) protein cingulin maintains apical surface stiffness and TJ membrane tortuosity and down-regulates apico-lateral membrane tension in MDCK cells. KO of cingulin in MDCK, mCCD and Eph4 cells results in a decrease in the juxta-membrane accumulation of labeling for cytoplasmic myosin-2B (NM2B), {gamma}-actin, phalloidin and ARHGEF18, but no detectable effect on myosin-2A (NM2A) and {beta}-actin. Loss of paracingulin leads to weaker mechanical phenotypes in MDCK cells, correlating with no detectable effect on the junctional accumulation of myosins and actins. Cingulin and paracingulin form biomolecular condensates, bind to the ZU5 domain of ZO-1, and are recruited as clients into ZO-1 condensates in a ZU5-dependent manner. Cingulin binding to ZO-1 promotes the unfolding of ZO-1, as determined by interaction with DbpA in cells lacking ZO-2 and in vitro. Cingulin promotes the accumulation of a pool of ZO-1 at the TJ and is required in a ZU5-dependent manner for the recruitment of phalloidin-labelled actin filaments into ZO-1 condensates, suggesting that ZU5-cingulin interaction promotes ZO-1 interaction with actin filaments. Our results indicate that cingulin tethers the juxta-membrane and apical branched {gamma}-actin-NM2B network to TJ to modulate ZO-1 conformation and the TJ assembly of a pool of ZO-1 and fine-tune the distribution of forces to apical and TJ membranes.

cell biology↗

bioRxiv2017

Tension-dependent stretching and folding of ZO-1 controls the localization of its interactors

Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays we show that the tight junction protein ZO-1 undergoes actomyosin tension-dependent stretching and folding in vivo. Magnetic tweezers experiments using purified ZO-1 indicate that pN-scale tensions (~2-4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact. Actomyosin tension and substrate stiffness regulate the localization and expression of the transcription factor DbpA and the tight junction membrane protein occludin in a ZO-1/ZO-2-dependent manner, resulting in modulation of gene expression, cell proliferation, barrier function and cyst morphogenesis. Interactions between the N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, and folding is antagonized by heterodimerization with ZO-2. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to modulate their protein interactions and downstream signaling.

cell biology↗