The distinct localization of CDC42 isoforms is responsible for their specific functions during migration.

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), which only differ in their carboxy-terminal sequence, including the CAAX motif essential for their association with membranes. We show that these divergent sequences do not directly affect the range of CDC42's potential binding partners but indirectly influence CDC42-driven signaling by controlling the subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u associates with the leading-edge plasma membrane of migrating cells, where it recruits the Par6-PKCζ complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it regulates N-WASP-mediated endocytosis. Both CDC42 isoforms contribute their specific functions to promote the chemotaxis of neural precursors, demonstrating that their expression pattern is decisive for tissue-specific cell behavior.

A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ-binding motif to the cytoplasm.

The carboxy-terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work, we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by E C-terminal specific antibodies nor by the PDZ domain-containing protein-binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.

SCRIB controls apical contractility during epithelial differentiation.

Although mutations in the SCRIB gene lead to multiple morphological organ defects in vertebrates, the molecular pathway linking SCRIB to organ shape anomalies remains elusive. Here, we study the impact of SCRIB-targeted gene mutations during the formation of the gut epithelium in an organ-on-chip model. We show that SCRIB KO gut-like epithelia are flatter with reduced exposed surface area. Cell differentiation on filters further shows that SCRIB plays a critical role in the control of apical cell shape, as well as in the basoapical polarization of myosin light chain localization and activity. Finally, we show that SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 and identify an evolutionary conserved SHROOM binding site in the SCRIB carboxy-terminal that is required for SCRIB function in the control of apical cell shape. Our results demonstrate that SCRIB plays a key role in epithelial morphogenesis by controlling the epithelial apical contractility during cell differentiation.

The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia.

BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-ß signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain. RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-ß signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding. CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.

Interactions of Severe Acute Respiratory Syndrome Coronavirus 2 Protein E With Cell Junctions and Polarity PSD-95/Dlg/ZO-1-Containing Proteins.

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ-binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains, which is identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified 10 human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, and LNX2) and MPP5/PALS1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2, and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4, and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in the E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

Microtubules tune mechanosensitive cell responses.

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.

Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and astrocyte migration.

Microtubules play a crucial role in mesenchymal migration by controlling cell polarity and the turnover of cell adhesive structures on the extracellular matrix. The polarized functions of microtubules imply that microtubules are locally regulated. Here, we investigated the regulation and role of two major tubulin post-translational modifications, acetylation and detyrosination, which have been associated with stable microtubules. Using primary astrocytes in a wound healing assay, we show that these tubulin modifications are independently regulated during cell polarization and differently affect cell migration. In contrast to microtubule detyrosination, αTAT1 (ATAT1)-mediated microtubule acetylation increases in the vicinity of focal adhesions and promotes cell migration. We further demonstrate that αTAT1 increases focal adhesion turnover by promoting Rab6-positive vesicle fusion at focal adhesions. Our results highlight the specificity of microtubule post-translational modifications and bring new insight into the regulatory functions of tubulin acetylation.This article has an associated First Person interview with the first author of the paper.

Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts.

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell-cell interactions.

Spectrin binding motifs regulate Scribble cortical dynamics and polarity function.

The tumor suppressor protein Scribble (SCRIB) plays an evolutionary conserved role in cell polarity. Despite being central for its function, the molecular basis of SCRIB recruitment and stabilization at the cell cortex is poorly understood. Here we show that SCRIB binds directly to the CH1 domain of ß spectrins, a molecular scaffold that contributes to the cortical actin cytoskeleton and connects it to the plasma membrane. We have identified a short evolutionary conserved peptide motif named SADH motif (SCRIB ABLIMs DMTN Homology) which is necessary and sufficient to mediate protein interaction with ß spectrins. The SADH domains contribute to SCRIB dynamics at the cell cortex and SCRIB polarity function. Furthermore, mutations in SCRIB SADH domains associated with spina bifida and cancer impact the stability of SCRIB at the plasma membrane, suggesting that SADH domain alterations may participate in human pathology.

APC binds intermediate filaments and is required for their reorganization during cell migration.

Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.

Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A.

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with ß-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding.

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.

Differential distribution of harmonin isoforms and their possible role in Usher-1 protein complexes in mammalian photoreceptor cells.

PURPOSE: Human Usher syndrome is the most common form of combined deafness and blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction, and prepubertal onset retinitis pigmentosa. Previous studies have shown that the USH1-proteins myosin VIIa, harmonin, and cadherin 23 interact and form a functional network during hair cell differentiation in the inner ear. The purpose of the present study was to analyze the molecular and cellular functions of these USH1 proteins in the mammalian retina. METHODS: Antibodies to USH1 proteins were generated and used in Western blot analysis of subcellular photoreceptor fractions and immunofluorescence and electron microscopy of the retina. RESULTS: Splice variants of harmonin were differentially expressed in the photoreceptor cell compartments. Whereas harmonin b isoforms were restricted to the light-sensitive outer segment, the harmonin a and c isoforms were more ubiquitously distributed in the photoreceptors. At the synaptic terminal of photoreceptor cells, harmonin a and c colocalized with myosin VIIa and cadherin 23. CONCLUSIONS: USH1 molecules can assemble to a supramolecular complex at photoreceptor synapses. Such a complex may contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions, which are thought to play a fundamental role in the organization of synaptic junctions. Dysfunction of any of the USH1 complex partners may lead to synaptic dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of patients with USH1. Furthermore, in photoreceptor outer segments, harmonin may also contribute to the clustering of outer segment proteins into supramolecular complexes.

Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.