Journal of Cell Science

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

The Golgi is a dynamic organelle with a unique morphology that has implications on its function. How the structural integrity of the Golgi is maintained despite its dynamic nature has been a long-standing question. Several siRNA-based screens have addressed this question and have identified a number of key players required for Golgi integrity. Interestingly, they also reported heterogeneity of phenotypic responses with regards to Golgi morphology. Although never systematically investigated, this variability has generally been attributed to poor transfection efficiency or cell cycle specific responses. Here we show that this heterogeneity is the result of differential response to the siRNA knockdown in different Golgi phenotypes, independent of transfection efficiency or cell cycle phases. To characterize the observed Golgi phenotype-specific responses at the molecular level we have developed an automated assay which enables microscopy-based phenotype classification followed by phenotype-specific single-cell transcriptome analysis. Application of this novel approach to the siRNA mediated knockdown of USO1, a key trafficking protein at the ER to Golgi boundary, surprisingly suggests a key involvement of the late endosomal/endocytic pathways in the regulation of Golgi organization. Our pipeline is the first of its kind developed to study Golgi organization, but can be applied to any biological problem that stands to gain from correlating morphology with single-cell readouts. Moreover, its automated and modular nature allows for uncomplicated scaling up, both in throughput and in complexity, helping the user achieve a systems level understanding of cellular processes.

Cells sense and respond to mechanical forces through mechanotransduction, which regulates processes in health and disease. In single cells, mechanotransduction involves the transmission of force to the cell nucleus, where it affects nucleocytoplasmic transport (NCT) and the subsequent nuclear localization of transcriptional regulators such as YAP. However, if and how NCT is mechanosensitive in multicellular systems is unclear. Here, we characterize and use a fluorescent sensor of nucleocytoplasmic transport (Sencyt) and demonstrate that nucleocytoplasmic transport responds to mechanics but not cell density in cell monolayers. Using monolayers of both epithelial and mesenchymal phenotype, we show that NCT is altered in response both to osmotic shocks, and to the inhibition of cell contractility. Further, NCT correlates with the degree of nuclear deformation measured through nuclear solidity, a shape parameter related to nuclear envelope tension. In contrast and in opposition to YAP, NCT is not affected by cell density, showing that the response of YAP to both mechanics and cell-cell contacts operates through distinct mechanisms. Our results demonstrate the generality of the mechanical regulation of NCT.

The recycling of integrin endocytosed during focal adhesion (FA) disassembly is critical for cell migration and contributes to the polarized formation of new FAs toward the leading edge. How this occurs is unclear. Here, we sought to identify the kinesin motor protein(s) that is involved in recycling endocytosed integrin back to the plasma membrane. We show that the kinesin-2 heterodimer, KIF3AC and the Rab11 adaptor protein RCP are required for FA reformation after the disassembly of FAs in mouse and human fibroblasts. In the absence of KIF3AC, integrin does not return to the cell surface after FA disassembly and is found in the Rab11 endocytic recycling compartment. Biochemical pulldowns revealed that KIF3C associated with {beta}1 integrin in an RCP dependent fashion, but only after FA disassembly. KIF3AC knockdown inhibited cell migration, trafficking of RCP toward the leading edge, and polarized formation of FAs at the leading edge. These results show that KIF3AC promotes cell migration by recycling integrin so that it generates new FAs in a polarized fashion. SummaryThe study reveals that the heterodimeric kinesin-2 motor KIF3AC and its adaptor RCP are crucial for polarized formation of focal adhesions at the front of migrating fibroblasts. KIF3AC and RCP associate with intracellularly recycling integrin to promote its return to the cell surface after its endocytosis from disassembled focal adhesions.

Gap junction communication is reduced during mitosis as the junction protein connexin-43 (Cx43) is redistributed from gap junction plaques on the plasma membrane to cytoplasmic annular vesicles and actin-based mitotic nanotubes that transiently connect mitotic cells to neighboring cells. However, the dynamic details of Cx43 redistribution during cell entry into and exit from mitosis, and the roles of mitotic nanotubes and associated Cx43 in intercellular communication, remain poorly understood. Here, using confocal live-cell imaging, we show that as cells enter mitosis, plaque-derived Cx43 structures are transferred to mitotic nanotubes. Over time, these structures fragment and migrate along the length of the nanotubes, either being transferred to the cytoplasm of adjacent cells or being positioned at the nanotube ends where they could potentially enable communication. Functionally, mitotic nanotubes indeed facilitate gap junction-dependent intercellular communication, though at reduced rates compared interphase cells. Interestingly, knockdown of Cx43 resulted in impaired nanotube formation and intercellular communication while inhibition of Rho kinase (ROCK) with Y-27632 prevented mitotic cell rounding and nanotube elongation, and increased cell-cell communication during mitosis, suggesting that nanotube function is influenced by Cx43 expression and trafficking as well as actin remodeling via ROCK. Overall, these findings provide valuable insights into the mechanisms that regulate Cx43 and mitotic nanotube dynamics and reveal a novel role for mitotic nanotubes in facilitating cell-cell communication during cell division.

Cell migration is a fundamental process pertaining to many critical physiological events. The ability to form and release adhesion structures is necessary for cell migration. The Calpain family of cysteine proteases are known to target adhesion proteins as their substrates and modulate adhesion dynamics. The two best studied Calpains, Calpain 1 and Calpain 2 form catalytically active holoenzymes through heterodimerization with a common non-catalytic regulatory small subunit known as Calpain 4. In previous studies, we determined that calpains are important in the production of traction forces and in the sensing of localized mechanical stimulation from the external environment. We found that perturbation of either Calpain 1 or 2 had no effect on the generation of traction forces. However, traction forces were weak when Calpain 4 was silenced. On the other hand, silencing of Calpain 1, 2, or 4 resulted in deficient sensing of external mechanical stimuli. These results together suggest that Calpain 4 functions independent of the catalytic large subunits in the generation of traction forces but functions together with either catalytic subunit in sensing external mechanical stimuli. The small subunit Calpain 4 contains 268 a.a. and is composed of 2 domains, the N-terminal domain V and C-terminal domain VI. Domain VI is a calmodulinlike domain containing five consecutive EF-hand motifs, of which the fifth one heterodimerizes with a large subunit. Moreover, domain V contains the common sequence GTAMRILGGVI that suggests cell membrane interactions. Given these attributes of domain V and VI of Calpain 4, we speculated that an individual domain might provide the functional properties for either traction or sensing. Therefore, each domain was cloned and expressed individually in Capn4-/- cells and assayed for traction and sensing. Results revealed that over-expression of domain V was sufficient to rescue the traction forces defect in Capn4-/- cells while overexpression of domain VI did not rescue the traction force. Consistent with our hypothesis, overexpression of domain VI rescued the sensing defect in Capn4-/- cells while overexpression of domain V had no effect. These results suggest that individual domains of Calpain 4 do indeed function independently to regulate either traction force or the sensing of external stimuli. We speculate that membrane association of Calpain 4 is required for the regulation of traction force and its association with a catalytic subunit is necessary for mechanosensing.

The site of nucleation strongly determines microtubule organisation and dynamics. The centrosome is a primary site for microtubule nucleation and organisation in most animal cells. In recent years, the Golgi apparatus has emerged as a site of microtubule nucleation and stabilisation. The microtubules originating from Golgi are essential for maintaining Golgi integrity post-Golgi trafficking, establishing cell polarity and enabling cell motility. Although the mechanism of nucleation and functional relevance of the Golgi-nucleated microtubule is well established, its regulation needs to be better studied. In this study, we report that DNA damage leads to aberrant Golgi structure and function accompanied by reorganisation of the microtubule network. Characterisation of microtubule dynamics post DNA damage showed the presence of a stable pool of microtubules resistant to depolymerisation by nocodazole and enriched in acetylated tubulin. Investigation of the functional association between Golgi dispersal and microtubule stability revealed that the Golgi elements were distributed along the acetylated microtubules. Microtubule regrowth assays showed an increase in Golgi-derived microtubule post DNA damage. Interestingly, reversal of Golgi dispersal reduces microtubule stabilisation. Altered intracellular trafficking resulting in mislocalisation of cell-cell junction proteins was observed post DNA damage. We propose that the increase in stable microtubules deregulates intracellular trafficking, resulting in cell polarity changes. This study would thus be the first to demonstrate the link between Golgi dispersal and microtubule reorganisation orchestrating changes in cell polarity.

P21 activated kinases (PAKs) are involved in a wide range of functions from the regulation of the cytoskeleton to the control of apoptosis and proliferation. Although many PAK substrates identified are implicated in the regulation of the actin cytoskeleton, a coherent picture of the total effect of PAK activation on the state of the actin cytoskeleton is unclear. We therefore set out to observe and quantify the effect of PAK inhibition on the actin cytoskeleton in greater detail. In Mouse Embryonic Fibroblasts, inhibition of PAK kinase activity, either by treatment with small molecule inhibitors or overexpression of mutant PAK constructs leads to the constitutive production of patches of the phosphoinositide PIP3 on the ventral surface of the cell. The formation of these patches remodels the actin cytoskeleton and polarises the cell. From the overexpression of truncated and mutant PAK constructs as well as an in vitro model of PAK recruitment to small GTPases we propose that this is due to a hyper recruitment of PAK and PAK binding partners in the absence of PAK kinase activity. This aberrant production of PIP3 suggests that, by limiting its own recruitment, the kinase activity of class I PAKs acts to downregulate PI3K activity, further highlighting class I PAKs as regulators of PI3K activity and therefore the excitability of the actin cytoskeleton.

The small GTPase Rho5 acts as a central hub to mediate the yeasts response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane, via fusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interaction in vivo. We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cell stiffness, and specific responses to features of the underlying substratum. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking the clathrin heavy chain (Cltc), display higher Youngs modulus, indicative of greater cellular stiffness, in comparison to WT mESCs. We have previously shown that mESCs lacking Cltc display a loss of pluripotency, and an initiation of differentiation. The increased stiffness observed in these cells was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein, Cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Youngs modulus, to values similar to those obtained with WT mESCs. However, the expression profile of pluripotency factors was not rescued. This indicates that a restoration of mechanical properties, through modulation of the actin cytoskeleton, may not always be accompanied by a change in the expression of critical transcription factors that regulate the state of a stem cell, and that this may be dependent on the presence of active endocytosis in a cell.

Protein kinase C epsilon (PKC{varepsilon}), a diacylglycerol (DAG)/phorbol ester-regulated PKC isoform, has been widely linked to oncogenesis and metastasis. PKC{varepsilon} plays important roles in the regulation of motility and invasiveness in non-small cell lung cancer (NSCLC). We previously reported that this kinase becomes prominently down-regulated upon TGF-{beta}-induced epithelial-to-mesenchymal transition (EMT), which leads to prominent phenotypic changes. While the phorbol ester PMA causes down-regulation of PKC, {delta} and {varepsilon} within hours, TGF-{beta} requires at least 4 days to reduce the expression levels of PKC{varepsilon} without affecting the expression of other PKCs, an effect that parallels the acquisition of a mesenchymal phenotype. Despite the prominent transcriptional component involved in EMT, we found that PKC{varepsilon} down-regulation does not involve changes in PKC{varepsilon} mRNA levels and was entirely independent of transcriptional activation of the PRKCE gene. Further mechanistic analysis revealed that the reduction in PKC{varepsilon} expression is dependent on proteasomal and endolysosomal pathways, but independent of autophagy processing mechanisms. Site-directed mutagenesis of Lys312 and Lys321 in PKC{varepsilon} prevented its down-regulation in response to either TGF-{beta} or the phorbol ester PMA. The shift in PKC{varepsilon} isozyme levels depending on cell plasticity underscores relevant functional consequences by modulating the expression of this oncogenic/metastatic kinase and highlights key roles of protein stability mechanisms in the control of PKC{varepsilon} phenotypic outcomes.

The Golgi apparatus comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region of most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi apparatus with intermediate filaments. In this study we show that the Golgi is in close physical proximity to vimentin intermediate filaments (IFs) in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with IFs. Although loss of vimentin and/or GORAB does not have major effects upon Golgi morphology at steady-state, the Golgi undergoes more rapid disassembly upon chemical disruption with the drug brefeldin A, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin causes reduced Golgi ribbon integrity when cells are cultured on high stiffness hydrogels, which is exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi apparatus, and suggest a role for GORAB in this process.

Cofilin acts as a key regulator of actin cytoskeletal remodeling via stimulating actin filament disassembly. Cofilin is inactivated by Ser-3 phosphorylation and reactivated by cofilin-phosphatase Slingshot-1 (SSH1). SSH1 is activated upon binding to F-actin, and this activation is inhibited by its phosphorylation at Ser-937 and Ser-978 and the subsequent binding of 14-3-3 proteins. In this study, we identified MARK3 (also named Par-1a and C-TAK1) as a kinase responsible for Ser-937/Ser-978 phosphorylation of SSH1. MARK3-mediated phosphorylation promoted SSH1 binding to 14-3-3 proteins and suppressed its F-actin-assisted cofilin-phosphatase activity. When Jurkat cells were stimulated with SDF-1, actin filaments formed multidirectional F-actin-rich lamellipodia around the cells in the initial stage, and thereafter, they were rearranged as a single polarized lamellipodium to the direction of cell migration. Upon SDF-1 stimulation, SSH1 was translocated into F-actin-rich lamellipodia, but its Ser-937/Ser-978 non-phosphorylatable mutant SSH1(2SA) was retained at the location of the original cortical F-actin. Knockdown of MARK3 or overexpression of SSH1(2SA), similar to SSH1 knockdown, impaired the conversion of multiple lamellipodia to a single polarized lamellipodium. These results indicate that MARK3-mediated Ser-937/Ser-978 phosphorylation is required for SSH1 liberation from F-actin and translocation to lamellipodia, and hence, facilitates the formation of a single polarized lamellipodium for directional cell migration. Our results suggest that the phosphorylation-dephosphorylation cycle of SSH1 is crucial for its localization to lamellipodia via promoting the dissociation-reassociation cycle of SSH1 to F-actin, and thereby the stimulus-induced lamellipodium formation to the direction of cell movement.

Traction force and mechanosensing (the ability to sense the mechanical attributes of the environment) are two key factors that enable a cell to modify its behavior during migration. Previously, it was determined that the calpain small subunit, calpain 4 (CapnS1), regulates the production of traction force independent of its proteolytic holoenzyme. A proteolytic enzyme is formed by calpain 4 binding to either of its catalytic partners, calpain 1 and 2. To further understand how calpain 4 regulates traction force, we used two-hybrid analysis to identify more components of the traction pathway. We discovered that basigin, an integral membrane protein and a documented inducer of matrix-metalloprotease (MMP), binds to calpain 4 in two-hybrid and pull-down assays. Traction force was deficient when basigin was silenced in MEF cells, and this deficiency was also reflected in the defect in substrate adhesion strength. Unlike Capn4-/- MEF cells, the cells deficient in basigin had normal mechanosensing abilities. Together, these results implicate basigin in the pathway in which calpain 4 regulates traction force independent of the catalytic large subunits.

Adherens junctions (AJs) are E-cadherin-based adhesions at cell-cell contacts that connect the actin cytoskeleton of epithelial cells. Formation and maturation of these junctions is important in development, e.g. for the generation of epithelial tissues, and loss of adherens junctions is linked to metastasis in cancer. It is well established that AJ formation is a mechano-sensitive process involving cis and trans clustering of E-cadherin and the mechanotransducive activation of -catenin that connects E-cadherin with the actin cytoskeleton. However, how mobility of E-cadherin in the cell membrane and their local density influences actin polymerisation is less well understood due to limitations in controlling physical properties of cell membranes and performing high-resolution microscopy in model organisms or cell-monolayers. Here we have created a biomimetic system enabling super-resolution microscopy of AJs by placing MCF7 cells, labelled with fluorescent actin, E-cadherin and -catenin, on fluid supported lipid bilayers (SLB) containing the extracellular domain of E-cadherin. We found that MCF7 cells were able to attach and spread on these substrates, recruiting E-cadherin and -catenin to form AJs that can mature and are mobile. Interestingly, we found that depending on E-cadherin mobility within the SLB, distinct types of actin architecture emerge. Low mobility substrates support formin-based linear actin polymerisation while high mobility substrates support Arp2/3-based branched actin network polymerisation. These polymerising actin structures are spatially confined to regions of low E-cadherin density suggesting they may play a role in AJ repair. Following how these actin structures at the cell-SLB interface evolve over time indicates regions of both linear and branched actin being present at mature cell-cell contacts.

Integrin-mediated cell-matrix adhesion regulates cell growth and survival and is often deregulated in transformed cancer cells, promoting anchorage independence. Integrins are essential regulators of cellular mechanotransduction, known to be altered in cancer cells. In breast cancers, tumour stiffening is a well-characterized feature of the disease progression. The Golgi apparatus, regulated by integrin-mediated adhesion in responding to mechanosensory cues, could support cancer progression. MDAMB231 and MCF7 cells with distinct Golgi organization and altered mechanosensing were evaluated. In MDAMB231 cells the organized Golgi in stable adherent cells becomes disorganised on loss of adhesion. This contrasts with MCF7 cells, where the Golgi remains disorganized regardless of the adhesion status. In MDAMB231 cells, increasing matrix stiffness promotes Golgi organization and regulates Golgi-dependent microtubule acetylation. In MCF7 cells, the Golgi stays disorganised despite increasing stiffness. Both cell types show stiffness-dependent cell spreading, with MCF7 cells spreading more efficiently than MDAMB231 cells - a difference that may be partially mediated by their differential Golgi organization. AXL, a receptor tyrosine kinase, is known to be involved in rigidity sensing and is differentially expressed in MDAMB231 (high) vs MCF7 (no expression) cells. AXL inhibition (by R428) and siRNA-mediated AXL knockdown disrupt stiffness-dependent Golgi organization in MDAMB231 cells, promoting cell spreading. Transient and stable AXL expression in MCF7 cells causes the Golgi to become predominantly organised and respond to higher matrix stiffness, affecting cell spreading. Stiffness-dependent AXL expression supports stiffness-dependent Arf1 expression and activation to drive Golgi organization. Thus, matrix mechanosensing through the AXL-Arf1-Golgi pathway could regulate vital cellular processes in breast cancer cells.

The interplay between cells and their surrounding microenvironment drives multiple cellular functions, including migration, proliferation, and cell fate transitions. The nucleus is a mechanosensitive organelle that adapts external mechanical and biochemical signals provided by the environment into nuclear changes with functional consequences for cell biology. However, the morphological and functional changes of the nucleus induced by 3D extracellular signals remain unclear. Here, we demonstrated that cells derived from 3D conditions conserve changes from cell confinement and show an aberrant nuclear morphology and localization of lamin B1, even in the absence of cellular confinement. We found that actin polymerization and protein kinase C (PKC) activity mediate the abnormal distribution of lamin B1 in 3D conditions-derived cells. These cells present altered chromatin compaction, gene transcription and cellular functions such as cell viability and migration. By combining biomechanical techniques and single-nucleus analysis, we have determined that the nucleus from 3D conditions-derived cells shows a different mechanical behavior and biophysical signature than the nucleus from control cells. Together, our work substantiates novel insights into how the extracellular environment alters the cell biology by promoting permanent changes in the chromatin, morphology, lamin B1 distribution, and the mechanical response of the nucleus.

Centrosomal and microtubule-associated proteins such as CEP170 and WDR62 are essential in regulating mitotic spindle formation and pole orientation during cell division. MAPKBP1, a paralog of WDR62, is also a centrosomal protein, but its function is currently unclear. We have shown here that MAPKBP1 is localised to the subdistal appendages of the mother centriole, the pericentriolar material (PCM) of the centrosomes and the mitotic spindles during metaphase. Furthermore, MAPKBP1, WDR62 and CEP170 exists as a complex, where MAPKBP1 is recruited to the centrosomes by WDR62 and CEP170, and CEP170-MAPKBP1 interaction is mediated by WDR62. In addition, MAPKBP1 depletion leads to mitotic spindle defects and delayed mitosis that were further exacerbated with WDR62 knockout, indicating a possible redundancy between MAPKBP1 and WDR62. MAPKBP1 loss also leads to PCM fragmentation, which supports its role as a subdistal appendages protein vital in maintaining centrosome structure and PCM cohesion for proper anchoring of mitotic spindles. This study provides insight into how subdistal appendages and centrosome and microtubule associated proteins co-operate to tightly regulate mitotic spindle formation and stability.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear if these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

Tubulin post-translational modifications regulate microtubule dynamics; among these, -tubulin acetylation has been linked to microtubule stability. We generated a Chlamydomonas mutant lacking the acetyltransferase TAT1, which completely abolished -tubulin K40 acetylation. Surprisingly, the lengths of normally acetylated structures, axonemes and rootlets, were largely unaffected. TAT1 localized to the flagellar tip, suggesting that it is the primary site of acetylation. Loss of acetylation caused an increase in axonemal tubulin turnover, as revealed by dikaryon-fusion assays. Unexpectedly, the tat1 mutant displayed an increased number of dynamic cytoplasmic microtubules and could regenerate long flagella after amputation, even when protein synthesis was inhibited. Despite these cytoskeletal changes, steady-state flagellar length, cell growth, and cell division remained essentially normal. These findings suggest that acetylation modulates microtubule behavior by regulating axonemal tubulin turnover and cytoplasmic microtubule dynamics, while cellular morphology is buffered against variations in microtubule content.