Susanne Bechstedt

Ciliogenesis is an evolutionarily conserved process that leads to the assembly of cilia. This process relies on microtubule-associated proteins (MAPs) to regulate axonemal microtubule dynamics. Misregulation of MAPs often leads to changes in ciliary homeostasis, contributing to numerous ciliopathies. Although CKAP2 and CKAP2-Like are best known for regulating microtubule dynamics during cell division, they are also components of ciliary organelles. Notably, CKAP2 overexpression promotes chromosomal instability and cancer, whereas CKAP2-Like deficiency causes Filippi syndrome, a developmental disorder with clinical features overlapping those of ciliopathies. How both MAPs operate at primary cilia remains poorly understood. Here, we identify both MAPs as axonemal components of motile and primary cilia in human cells. We find that CKAP2-positive cilia are linked to cell cycle progression and that the conserved intrinsically disordered microtubule-binding domain of CKAP2, which is essential for microtubule polymerization and stabilization, is required for its ciliary localization. Unexpectedly, CRISPR-mediated loss of either paralog results in elongated cilia without affecting ciliogenesis, accompanied by increased ciliary enrichment of the remaining paralog and impaired Gli2 accumulation at the ciliary tip. In contrast, simultaneous deletion of both MAPs suppresses ciliogenesis and has minimal effect on the ciliary length as compared to wild-type levels. These findings uncover that CKAP2 and CKAP2-Like are axonemal MAPs that contribute to ciliogenesis, partially compensating for each other in human cells.

Ciliogenesis is an evolutionarily conserved process that leads to the assembly of cilia. This process relies on microtubule-associated proteins (MAPs) to regulate axonemal microtubule dynamics. Misregulation of MAPs often leads to changes in ciliary homeostasis, contributing to numerous ciliopathies. Although CKAP2 and CKAP2-Like are best known for regulating microtubule dynamics during cell division, they are also components of ciliary organelles. Notably, CKAP2 overexpression promotes chromosomal instability and cancer, whereas CKAP2-Like deficiency causes Filippi syndrome, a developmental disorder with clinical features overlapping those of ciliopathies. How both MAPs operate at primary cilia remains poorly understood. Here, we identify both MAPs as axonemal components of motile and primary cilia in human cells. We find that CKAP2-positive cilia are linked to cell cycle progression and that the conserved intrinsically disordered microtubule- binding domain of CKAP2, which is essential for microtubule polymerization and stabilization, is required for its ciliary localization. Unexpectedly, CRISPR-mediated loss of either paralog results in elongated cilia without affecting ciliogenesis, accompanied by increased ciliary enrichment of the remaining paralog and impaired Gli2 accumulation at the ciliary tip. In contrast, simultaneous deletion of both MAPs suppresses ciliogenesis and has minimal effect on the ciliary length as compared to wild-type levels. These findings uncover that CKAP2 and CKAP2-Like are axonemal MAPs that contribute to ciliogenesis, partially compensating for each other in human cells.

The cerebral cortex is a multi-layered structure generated through the migration of neural precursors from their birthplace in the ventricular zone to their destination within the cortical plate. Neuronal migration defects are responsible for many human pathologies collectively called neuronal migration disorders, which include subcortical band heterotopia and cobblestone brain (COB) malformation. One example of a protein involved in a neuronal migration disorder is the echinoderm microtubule-associated protein-like 1 (EML1) protein, one of six members of the mammalian EML family. Absence of EML1 protein results in subcortical band heterotopia in mice and humans. Here, we report that the absence of the paralogous protein EML3 leads to delayed embryonic development and small size, and a COB-like phenotype with neuronal ectopias in the dorsal telencephalon. We found that EML3 is expressed in the neuroepithelium and meningeal mesenchyme when those tissues participate in pial basement membrane (PBM) formation. Transmission electron microscopy demonstrated that the extracellular matrix of the PBM is structurally abnormal in Eml3 null mice when the first radially migrating neurons arrive. The reduced structural integrity of the PBM leads to focal over-migration of neurons into the subarachnoid space. These findings strengthen the link between the EML protein family and cortical neuronal migration defects by identifying Eml3 as the first EML family member whose absence leads to over-migration of neuroblasts. Moreover, we report the first COB-like phenotype with PBM structural defects when a single microtubule-associated protein is deleted.

The cerebral cortex is a multi-layered structure generated through the migration of neural precursors from their birthplace in the ventricular zone to their destination within the cortical plate. Neuronal migration defects are responsible for many human pathologies collectively called neuronal migration disorders, which include subcortical band heterotopia and cobblestone brain (COB) malformation. One example of a protein involved in a neuronal migration disorder is the echinoderm microtubule-associated protein-like 1 (EML1) protein, one of six members of the mammalian EML family. Absence of EML1 protein results in subcortical band heterotopia in mice and humans. Here, we report that absence of the paralogous protein EML3 leads to delayed embryonic development and small size, and a COB-like phenotype with neuronal ectopias in the dorsal telencephalon. We found that EML3 is expressed in the neuroepithelium and meningeal mesenchyme when those tissues participate in pial basement membrane (PBM) formation. Transmission electron microscopy demonstrated that the extracellular matrix of the PBM is structurally abnormal in Eml3 null mice when the first radially migrating neurons arrive. The reduced structural integrity of the PBM leads to focal over-migration of neurons into the subarachnoid space. These findings strengthen the link between the EML protein family and cortical neuronal migration defects by identifying Eml3 as the first EML family member whose absence leads to over-migration of neuroblasts. Moreover, we report the first COB-like phenotype with PBM structural defects when a single microtubule-associated protein is deleted.

The biorientation-defective (BOD) protein family, comprising BOD1, BOD1L1 (BOD1-like 1), and BOD1L2, plays critical and diverse roles in fundamental cellular processes, including mitosis, DNA repair, neurological function, and metabolism. BOD1 and BOD1L2 are small proteins of less than 200 amino acids, whereas BOD1L1 contains a long C-terminal extension, totaling 3042 amino acids. BOD1 was originally identified in Xenopus laevis oocyte chromatin extracts. Subsequent work in mitotic human cells demonstrated that BOD1 is an outer kinetochore protein that regulates PP2A-B56 phosphatase function and consequently is vital for chromosome biorientation and segregation fidelity, hence the name. BOD1 also has important roles in neurological function and lipid metabolism as a component of the COMPASS (complex of proteins associated with SET1)–SETD1B complex. In contrast, BOD1L1 was first identified as a phosphorylated target of the ATM kinase and then highlighted in a screen for DNA replication fork components. Further work demonstrated a role for BOD1L1 in DNA double-stranded break repair, where BOD1L1 is required to recruit the protein RIF1 to damaged chromatin to enable efficient DNA repair and control sensitivity to radio/chemotherapeutics. Consistently, BOD1L1 binds known DNA damage/repair/replication proteins, including FANCD2, RIF1, and BRCA2. Loss of BOD1L1 causes catastrophic genome instability through misrepair and/or overprocessing of damaged DNA. Recently, BOD1L1 has also been shown to regulate the PP2A-B56 phosphatase at kinetochores in mitotic human cells. In contrast, little is known about BOD1L2, which is only expressed in sperm cells and precursors. In this review, we describe recent progress in understanding the functions of this protein family and discuss future avenues of research.

Summary: Faithful chromosome segregation requires the precise assembly of the mitotic spindle. Cytoskeleton-associated protein 2 (CKAP2) is a microtubule-associated protein with potent microtubule-polymerizing activity that localizes to spindle microtubules during mitosis. Loss or overexpression of CKAP2 causes chromosomal instability and aneuploidy, yet its regulation remains poorly understood. To identify CKAP2 interactors, we immunoprecipitated endogenous CKAP2 from mitotic RPE1 cells and analysed co-purifying proteins by mass spectrometry. This revealed a specific interaction between CKAP2 and the mitotic kinase Aurora A and its activator TPX2, but not with Aurora B, as previously suggested. We further show that CKAP2 co-localizes with Aurora A-TPX2 complexes throughout mitosis, and Aurora A directly phosphorylates CKAP2 in cells and in vitro. Phosphorylation of CKAP2 By Aurora kinase A decreases its microtubule affinity in cells and in vitro. These findings identify CKAP2 as a direct interactor of TPX2 and a substrate of Aurora kinase A and uncover a regulatory pathway controlling spindle growth and stability during mitosis.

Intrinsically disordered proteins (IDPs) challenge the traditional structure-function paradigm by lacking a stable three-dimensional structure ¹ . While their roles as dynamic effectors, scaffolds, and molecular switches are well-established, it has been widely accepted that enzymatic activity requires a stably folded catalytic center ² . Here, we challenge this dogma by demonstrating that a 284-amino acid intrinsically disordered domain of the cytoskeleton-associated protein 2 (CKAP2) is sufficient to catalyze both microtubule polymerization and depolymerization. CKAP2 promotes tubulin incorporation without high-affinity tubulin binding, suggesting a transition-state-based catalytic mechanism distinct from known microtubule polymerases. These findings establish, for the first time, that an intrinsically disordered domain can function as a bona fide enzyme, expanding our understanding of the functional repertoire of disordered proteins and their roles in cellular processes.

Maspin/SerpinB5 is an abundant and pleiotropic protein mostly expressed by epithelia. Initially described as a tumor suppressor, it has been reported as a regulator of cell adhesion, migration, and invasion. How intracellular Maspin orchestrates these processes is poorly understood. In this study, we utilized Affinity purification-Mass spectrometry (AP/MS) alongside in vitro reconstitution assays to establish that Maspin directly interacts with microtubules and microfilaments. Additionally, CRISPR/Cas9-mediated GFP tagging of endogenous Maspin, combined with immunostaining, revealed its localization at the cortical cytoskeleton and the mitotic spindle. Depletion of Maspin by RNAi and CRISPR/Cas9 in non-transformed epithelial cell lines modifies cell-cell contact and promotes cytoskeletal rearrangements. Concomitantly, we observed a modest upregulation of mesenchymal markers during interphase and abnormal cell rounding during mitosis. An evaluation of Maspin’s effect on microtubules revealed that it suppresses their growth in vitro and in cells. Collectively, these results demonstrate that Maspin acts dynamically at the interface of the cytoskeleton and adhesion sites, modulating cell shape.

The cerebral cortex is a multi-layered structure generated through the migration of neural precursors from their birthplace in the ventricular zone to their destination within the cortical plate. Neuronal migration defects are responsible for many human pathologies collectively called neuronal migration disorders, which include subcortical band heterotopia and cobblestone brain (COB) malformation. One example of a protein involved in a neuronal migration disorder is the echinoderm microtubule-associated protein-like 1 (EML1) protein, one of six members of the mammalian EML family. Absence of EML1 protein results in subcortical band heterotopia in mice and humans. Here, we report that absence of the paralogous protein EML3 leads to delayed embryonic development and small size, and a COB-like phenotype with neuronal ectopias in the dorsal telencephalon. We found that EML3 is expressed in the neuroepithelium and meningeal mesenchyme when those tissues participate in pial basement membrane (PBM) formation. Transmission electron microscopy demonstrated that the extracellular matrix of the PBM is structurally abnormal in Eml3 null mice when the first radially migrating neurons arrive. The reduced structural integrity of the PBM leads to focal over-migration of neurons into the subarachnoid space. These findings strengthen the link between the EML protein family and cortical neuronal migration defects by identifying Eml3 as the first EML family member whose absence leads to over-migration of neuroblasts. Moreover, we report the first COB-like phenotype with PBM structural defects when a single microtubule-associated protein is deleted.

Maspin/SerpinB5 is an abundant and pleiotropic protein mostly expressed by epithelia. Initially described as a tumor suppressor, it has been reported as a regulator of cell adhesion, migration, and invasion. How intracellular Maspin orchestrates these processes is poorly understood. In this study, we utilized Affinity purification-Mass spectrometry (AP/MS) alongside in vitro reconstitution assays to establish that Maspin directly interacts with microtubules and microfilaments. Additionally, CRISPR/Cas9-mediated GFP tagging of endogenous Maspin, combined with immunostaining, revealed its localization at the cortical cytoskeleton and the mitotic spindle. Depletion of Maspin by RNAi and CRISPR/Cas9 in three distinct epithelial cell lines disrupts cell-cell adhesion, reorganizes the cytoskeleton and results in upregulation of mesenchymal markers during interphase. In mitotic cells, loss of Maspin induces abnormal cell rounding and rearrangement of cortical F-actin. Moreover, Maspin suppresses microtubule growth in vitro and in cells. Collectively, these results demonstrate that Maspin acts at the interface between the cytoskeleton and adhesion sites, directly modulating cell shape and preventing epithelial-mesenchymal transition. Summary: Da Silva et al. report that the non-inhibitory serpin Maspin (SerpinB5), which has long been implicated in the regulation of cell adhesion, migration, invasion and metastasis, directly binds to microfilaments and microtubules in vitro and in cells, acting at the interface between the cytoskeleton and adhesion sites.