Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells.

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS-/- cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients.

De novo assembly and annotation of the CHOZN® GS-/- genome supports high-throughput genome-scale screening.

Chinese hamster ovary (CHO) cells have been used as the industry standard for the production of therapeutic monoclonal antibodies for several decades. Despite significant improvements in commercial-scale production processes and media, the CHO cell has remained largely unchanged. Due to the cost and complexity of whole-genome sequencing and gene-editing it has been difficult to obtain the tools necessary to improve the CHO cell line. With the advent of next-generation sequencing and the discovery of the CRISPR/Cas9 system it has become more cost effective to sequence and manipulate the CHO genome. Here, we provide a comprehensive de novo assembly and annotation of the CHO-K1 based CHOZN® GS-/- genome. Using this platform, we designed, built, and confirmed the functionality of a whole genome CRISPR guide RNA library that will allow the bioprocessing community to design a more robust CHO cell line leading to the production of life saving medications in a more cost-effective manner.

Mislocalization of cone nuclei impairs cone function in mice.

The nuclei of cone photoreceptors are located on the apical side of the outer nuclear layer (ONL) in vertebrate retinas. However, the functional role of this evolutionarily conserved localization of cone nuclei is unknown. We previously showed that Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) are essential for the apical migration of cone nuclei during development. Here, we developed an efficient genetic strategy to disrupt cone LINC complexes in mice. Experiments with animals from both sexes revealed that disrupting cone LINC complexes resulted in mislocalization of cone nuclei to the basal side of ONL in mouse retina. This, in turn, disrupted cone pedicle morphology, and appeared to reduce the efficiency of synaptic transmission from cones to bipolar cells. Although we did not observe other developmental or phototransduction defects in cones with mislocalized nuclei, their dark adaptation was impaired, consistent with a deficiency in chromophore recycling. These findings demonstrate that the apical localization of cone nuclei in the ONL is required for the timely dark adaptation and efficient synaptic transmission in cone photoreceptors.

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

Multiple Isoforms of Nesprin1 Are Integral Components of Ciliary Rootlets.

SYNE1 (synaptic nuclear envelope 1) encodes multiple isoforms of Nesprin1 (nuclear envelope spectrin 1) that associate with the nuclear envelope (NE) through a C-terminal KASH (Klarsicht/Anc1/Syne homology) domain (Figure 1A) [1-4]. This domain interacts directly with the SUN (Sad1/Unc84) domain of Sun proteins [5-7], a family of transmembrane proteins of the inner nuclear membrane (INM) [8, 9], to form the so-called LINC complexes (linkers of the nucleoskeleton and cytoskeleton) that span the entire NE and mediate nuclear positioning [10-12]. In a stark departure from this classical depiction of Nesprin1 in the context of the NE, we report here that rootletin recruits Nesprin1α at the ciliary rootlets of photoreceptors and identify asymmetric NE aggregates of Nesprin1α and Sun2 that dock filaments of rootletin at the nuclear surface. In NIH 3T3 cells, we show that recombinant rootletin filaments also dock to the NE through the specific recruitment of an ∼600-kDa endogenous isoform of Nesprin1 (Nes1600kDa) and of Sun2. In agreement with the association of Nesprin1α with photoreceptor ciliary rootlets and the functional interaction between rootletin and Nesprin1 in fibroblasts, we demonstrate that multiple isoforms of Nesprin1 are integral components of ciliary rootlets of multiciliated ependymal and tracheal cells. Together, these data provide a novel functional paradigm for Nesprin1 at ciliary rootlets and suggest that the wide spectrum of human pathologies linked to truncating mutations of SYNE1 [13-15] may originate in part from ciliary defects.

The role of retinol dehydrogenase 10 in the cone visual cycle.

Pigment regeneration is critical for the function of cone photoreceptors in bright and rapidly-changing light conditions. This process is facilitated by the recently-characterized retina visual cycle, in which Müller cells recycle spent all-trans-retinol visual chromophore back to 11-cis-retinol. This 11-cis-retinol is oxidized selectively in cones to the 11-cis-retinal used for pigment regeneration. However, the enzyme responsible for the oxidation of 11-cis-retinol remains unknown. Here, we sought to determine whether retinol dehydrogenase 10 (RDH10), upregulated in rod/cone hybrid retinas and expressed abundantly in Müller cells, is the enzyme that drives this reaction. We created mice lacking RDH10 either in cone photoreceptors, Müller cells, or the entire retina. In vivo electroretinography and transretinal recordings revealed normal cone photoresponses in all RDH10-deficient mouse lines. Notably, their cone-driven dark adaptation both in vivo and in isolated retina was unaffected, indicating that RDH10 is not required for the function of the retina visual cycle. We also generated transgenic mice expressing RDH10 ectopically in rod cells. However, rod dark adaptation was unaffected by the expression of RDH10 and transgenic rods were unable to use cis-retinol for pigment regeneration. We conclude that RDH10 is not the dominant retina 11-cis-RDH, leaving its primary function in the retina unknown.

Lamin B1 and lamin B2 are long-lived proteins with distinct functions in retinal development.

Lamin B1 and lamin B2 are essential building blocks of the nuclear lamina, a filamentous meshwork lining the nucleoplasmic side of the inner nuclear membrane. Deficiencies in lamin B1 and lamin B2 impair neurodevelopment, but distinct functions for the two proteins in the development and homeostasis of the CNS have been elusive. Here we show that embryonic depletion of lamin B1 in retinal progenitors and postmitotic neurons affects nuclear integrity, leads to the collapse of the laminB2 meshwork, impairs neuronal survival, and markedly reduces the cellularity of adult retinas. In stark contrast, a deficiency of lamin B2 in the embryonic retina has no obvious effect on lamin B1 localization or nuclear integrity in embryonic retinas, suggesting that lamin B1, but not lamin B2, is strictly required for nucleokinesis during embryonic neurogenesis. However, the absence of lamin B2 prevents proper lamination of adult retinal neurons, impairs synaptogenesis, and reduces cone photoreceptor survival. We also show that lamin B1 and lamin B2 are extremely long-lived proteins in rod and cone photoreceptors. OF interest, a complete absence of both proteins during postnatal life has little or no effect on the survival and function of cone photoreceptors.

Nrl-Cre transgenic mouse mediates loxP recombination in developing rod photoreceptors.

The developing mouse retina is a tractable model for studying neurogenesis and differentiation. Although transgenic Cre mouse lines exist to mediate conditional genetic manipulations in developing mouse retinas, none of them act specifically in early developing rods. For conditional genetic manipulations of developing retinas, a Nrl-Cre mouse line in which the Nrl promoter drives expression of Cre in rod precursors was created. The results showed that Nrl-Cre expression was specific to the retina where it drives rod-specific recombination with a temporal pattern similar to endogenous Nrl expression during retinal development. This Nrl-Cre transgene does not negatively impact retinal structure and function. Taken together, the data suggested that the Nrl-Cre mouse line was a valuable tool to drive Cre-mediated recombination specifically in developing rods.

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope: positioning nuclei and organizing synapses.

The nuclear envelope plays an essential role in nuclear positioning within cells and tissues. This review highlights advances in understanding the mechanisms of nuclear positioning during skeletal muscle and central nervous system development. New findings, particularly about A-type lamins and Nesprin1, may link nuclear envelope integrity to synaptic integrity. Thus synaptic defects, rather than nuclear mispositioning, may underlie human pathologies associated with mutations of nuclear envelope proteins.

A variant of Nesprin1 giant devoid of KASH domain underlies the molecular etiology of autosomal recessive cerebellar ataxia type I.

Nonsense mutations across the whole coding sequence of Syne1/Nesprin1 have been linked to autosomal recessive cerebellar ataxia Type I (ARCA1). However, nothing is known about the molecular etiology of this late-onset debilitating pathology. In this work, we report that Nesprin1 giant is specifically expressed in CNS tissues. We also identified a CNS-specific splicing event that leads to the abundant expression of a KASH-LESS variant of Nesprin1 giant (KLNes1g) in the cerebellum. KLNes1g displayed a noncanonical localization at glomeruli of cerebellar mossy fibers whereas Nesprin2 exclusively decorated the nuclear envelope of all cerebellar neurons. In immunogold electron microscopy, KLNes1g colocalized both with synaptic vesicles within mossy fibers and with dendritic membranes of cerebellar granule neurons. We further identified vesicle- and membrane-associated proteins in KLNes1g immunoprecipitates. Together, our results suggest that the loss of function of KLNes1g resulting from Nesprin1 nonsense mutations underlies the molecular etiology of ARCA1.

Temporal and tissue-specific disruption of LINC complexes in vivo.

Migration and anchorage of nuclei within developing and adult tissues rely on Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes). These macromolecular assemblies span the nuclear envelope and physically couple chromatin and nuclear lamina to cytoplasmic cytoskeletal networks. LINC complexes assemble within the perinuclear space through direct interactions between the respective evolutionary-conserved SUN and KASH domains of Sun proteins, which reside within the inner nuclear membrane, and Nesprins, which reside within the outer nuclear membrane. Here, we describe and validate a dominant-negative transgenic strategy allowing for the disruption of endogenous SUN/KASH interactions through the inducible expression of a recombinant KASH domain. Our approach, which is based on the Cre/Lox system, allows for the targeted disruption of LINC complexes in a wide array of mouse tissues or specific cell types thereof and bypasses the perinatal lethality and potential cell nonautonomous effects of current mouse models based on germline inactivation of genes encoding Sun proteins and Nesprins. For these reasons, this mouse model provides a useful tool to evaluate the physiological relevance of LINC complexes integrity during development and homeostasis in a wide array of mammalian tissues.

Nuclear envelope in nuclear positioning and cell migration.

Hauling and anchoring the nucleus within immobile or motile cells, tissues, and/or syncytia represents a major challenge. In the past 15 years, Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) have emerged as evolutionary-conserved molecular devices that span the nuclear envelope and provide interacting interfaces for cytoskeletal networks and molecular motors to the nuclear envelope. Here, we review the molecular composition of LINC complexes and focus on how their genetic alteration in vivo has provided a wealth of information related to the relevance of nuclear positioning during tissue development and homeostasis with a special emphasis on the central nervous system. As it may be relevant for metastasis in a range of cancers, the involvement of LINC complexes in migration of nonneuronal cells via its interaction with the perinuclear actin cap will also be developed.

Developmental regulation of linkers of the nucleoskeleton to the cytoskeleton during mouse postnatal retinogenesis.

Sun proteins and Nesprins are two families of proteins whose direct interactions across the nuclear envelope provide for the core of Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) that physically connect the nucleus interior to cytoskeletal networks. Whereas LINC complexes play essential roles in nuclear migration anchorage and underlie normal CNS development, the developmental regulation of their composition remains largely unknown. In this study, we examined the spatiotemporal expression of lamins, Sun proteins and Nesprins during postnatal mouse retinal development. Whereas retinal precursor cells mostly express B-type lamins, Sun1, and high molecular weight isoforms of Nesprins, post-mitotic retinal cells are characterized by a drastic downregulation of the latter, the expression of A-type lamins, and the strong induction of a specific isoform of Nesprin1 late in retinal development. Importantly, our results emphasize different spatiotemporal expression for Nesprin1 and Nesprin2 and further suggest an important role for KASH-less isoforms of Nesprin1 in the CNS. In conclusion, the transition from retinal precursor cells undergoing interkinetic nuclear migration to post-mitotic retinal cells undergoing nuclear translocation and/or anchorage is accompanied by a profound remodeling of LINC complexes composition. This remodeling may reflect different requirements of nuclear dynamics at different stages of CNS development.

LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina.

It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatial confinement of cone nuclei as well as a new mouse model to evaluate the pathological relevance of nuclear mispositioning.

BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures.

Cytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N-dynein-dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end-directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration.

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles - the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions.

Analyses of dynein heavy chain mutations reveal complex interactions between dynein motor domains and cellular dynein functions.

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies.

UnLINCing the nuclear envelope: towards an understanding of the physiological significance of nuclear positioning.

Appropriate tissue morphogenesis strictly requires the developmental regulation of different types of nuclear movements. LINC (linker of nucleoskeleton and cytoskeleton) complexes are macromolecular scaffolds that span the nuclear envelope and physically connect the nuclear interior to different cytoskeletal elements and molecular motors, thereby playing essential roles in nucleokinesis. Recent studies dedicated to the in vivo disruption of LINC complexes not only confirmed their widespread role in nuclear dynamics, but also led to a vigorous regain of interest in the physiological relevance of nuclear positioning within cells and syncitia. In the present paper, we review the results of LINC complex disruption in vivo across different organisms and the potential implications of observed phenotypes in human diseases.

Bringing KASH under the SUN: the many faces of nucleo-cytoskeletal connections.

The nucleus is the most prominent cellular organelle, and its sharp boundaries suggest the compartmentalization of the nucleoplasm from the cytoplasm. However, the recent identification of evolutionarily conserved linkers of the nucleoskeleton to the cytoskeleton (LINC) complexes, a family of macromolecular assemblies that span the double membrane of the nuclear envelope, reveals tight physical connections between the two compartments. Here, we review the structure and evolutionary conservation of SUN and KASH domain-containing proteins, whose interaction within the perinuclear space forms the "nuts and bolts" of LINC complexes. Moreover, we discuss the function of these complexes in nuclear, centrosomal, and chromosome dynamics, and their connection to human disease.