Septins as membrane influencers: direct play or in association with other cytoskeleton partners.

The cytoskeleton comprises three polymerizing structures that have been studied for a long time, actin microfilaments, microtubules and intermediate filaments, plus more recently investigated dynamic assemblies like septins or the endocytic-sorting complex required for transport (ESCRT) complex. These filament-forming proteins control several cell functions through crosstalks with each other and with membranes. In this review, we report recent works that address how septins bind to membranes, and influence their shaping, organization, properties and functions, either by binding to them directly or indirectly through other cytoskeleton elements.

Cytoskeleton and Associated Proteins: Pleiotropic JNK Substrates and Regulators.

This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.

Cdc42 and its BORG2 and BORG3 effectors control the subcellular localization of septins between actin stress fibers and microtubules.

Cell resistance to taxanes involves several complementary mechanisms, among which septin relocalization from actin stress fibers to microtubules plays an early role. By investigating the molecular mechanism underlying this relocalization, we found that acute paclitaxel treatment triggers the release from stress fibers and subsequent proteasome-mediated degradation of binder of Rho GTPases 2 (BORG2)/Cdc42 effector protein 3 (Cdc42EP3) and to a lesser extent of BORG3/Cdc42EP5, two Cdc42 effectors that link septins to actin in interphase cells. BORG2 or BORG3 silencing not only caused septin detachment from stress fibers but also mimicked the effects of paclitaxel by triggering both septin relocalization to microtubules and significant drug resistance. Conversely, BORG2 or BORG3 overexpression retained septins on actin fibers even after paclitaxel treatment, without affecting paclitaxel sensitivity. We found that drug-induced inhibition of Cdc42 resulted in a drop in BORG2 level and in the relocalization of septins to microtubules. Accordingly, although septins relocalized when overexpressing an inactive mutant of Cdc42, the expression of a constitutively active mutant acted locally at actin stress fibers to prevent septin release, even after paclitaxel treatment. These findings reveal the role of Cdc42 upstream of BORG2 and BORG3 in controlling the interplay between septins, actin fibers, and microtubules in basal condition and in response to taxanes.

Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue.

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170's capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.

Septin filament coalignment with microtubules depends on SEPT9_i1 and tubulin polyglutamylation, and is an early feature of acquired cell resistance to paclitaxel.

Cancer cell resistance to taxanes is a complex, multifactorial process, which results from the combination of several molecular and cellular changes. In breast cancer cells adapted to long-term paclitaxel treatment, we previously identified a new adaptive mechanism that contributes to resistance and involves high levels of tubulin tyrosination and long-chain polyglutamylation coupled with high levels of septin expression, especially that of SEPT9_i1. This in turn led to higher CLIP-170 and MCAK recruitment to microtubules to enhance microtubule dynamics and therefore counteract the stabilizing effects of taxanes. Here, we explored to which extent this new mechanism alone could trigger taxane resistance. We show that coupling septins (including SEPT9_i1) overexpression together with long-chain tubulin polyglutamylation induce significant paclitaxel resistance in several naive (taxane-sensitive) cell lines and accordingly stimulate the binding of CLIP-170 and MCAK to microtubules. Strikingly, such resistance was paralleled by a systematic relocalization of septin filaments from actin fibers to microtubules. We further show that this relocalization resulted from the overexpression of septins in a context of enhanced tubulin polyglutamylation and reveal that it could also be promoted by an acute treatment with paclitaxel of sensitve cell displaying a high basal level of SEPT9_i1. These findings point out the functional importance and the complex cellular dynamics of septins in the onset of cell resistance to death caused by microtubule-targeting antimitotic drugs of the taxane family.

Microtubule reorientation in the blue spotlight: Cutting and CLASPing at dynamic hot spots.

Microtubule reorientation into a longitudinal network during the phototropic response in Arabidopsis thaliana depends on their severing by katanin at crossovers. Lindeboom et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201805047) show that at newly generated plus ends, the anti-catastrophe activity of CLASP is essential for further growth.

PI(4,5)P2 produced by the PI4P5K SKTL controls apical size by tethering PAR-3 in Drosophila epithelial cells.

BACKGROUND: The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides is controlled by the concerted action of phosphoinositide kinases and phosphatases. RESULTS: Using the Drosophila follicular epithelium as a model system in vivo, we show here that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated by the PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, we observe the disassembly of adherens junctions, actin cytoskeleton reorganization, and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. We provide evidence that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, we show that PI(4,5)P2 is excluded from the cell apex and that PAR-3 diffuses laterally just prior to the apical constriction in a context of endogenous invagination. CONCLUSIONS: All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain.

Drosophila tubulin-binding cofactor B is required for microtubule network formation and for cell polarity.

Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-ß-tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and ß-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division.

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.

A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic toxicity of PABPN1.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.

An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila.

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.

Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila.

The Suppressor of forked [Su(f)] protein is the Drosophila homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3' end processing reaction in vitro. We have addressed directly the role of su(f) in the mRNA 3' end processing reaction in vivo. We show that su(f) is required for the cleavage of pre-mRNA during mRNA 3' end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the Drosophila protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3' end formation in su(f) mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of Drosophila. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of su(f) mutants cannot restore utilization of a regulated poly(A) site in Drosophila. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3' end formation in vivo, but that these two proteins are not interchangeable for regulation of poly(A) site utilization.