validation-mouse-model-to-disrupt-linc-complexes-cell-specific

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 ...

Motor-based intracellular transport and its regulation are crucial to the functioning of a cell. Disruption of transport is linked to Alzheimer's and other neurodegenerative diseases. However, many fundamental aspects of transport are poorly understood. An important issue is how cells achieve and regulate efficient long-distance transport. Mounting evidence suggests that many in vivo cargoes are transported along microtubules by more than one motor, but we do not know how multiple motors work together or can be regulated. Here we first show that multiple kinesin motors, working in conjunction, can achieve very long distance transport and apply significantly larger forces without the need of additional factors. We then demonstrate in vitro that the important microtubule-associated protein, tau, regulates the number of engaged kinesin motors per cargo via its local concentration on microtubules. This function of tau provides a previously unappreciated mechanism to regulate transport. By reducing motor reattachment rates, tau affects cargo travel distance, motive force, and cargo dispersal. We also show that different isoforms of tau, at concentrations similar to those in cells, have dramatically different potency. These results provide a well defined mechanism for how altered tau isoform levels could impair transport and thereby lead to neurodegeneration without the need of any other pathway.

Multiple-motor based transport and its regulation by Tau.

Motor-basierte intrazellul&auml;ren Transport und seine Verordnung sind entscheidend f&uuml;r das Funktionieren einer Zelle. St&ouml;rung des Verkehrs ist verkn&uuml;pft mit Alzheimer und anderen neurodegenerativen Erkrankungen. Allerdings sind viele grundlegende Aspekte des Verkehrswesens schlecht verstanden. Eine wichtige Frage ist, wie Zellen zu erreichen und effiziente Fernverkehr zu regulieren. Mehrenden Beweise zeigen, dass viele in-vivo-Ladungen werden durch mehr als ein Motor entlang der Mikrotubuli transportiert, aber wir wissen nicht, wie mehrere Motoren zusammenarbeiten oder reguliert werden k&ouml;nnen. Hier zeigen wir zuerst, dass mehrere Kinesin Motoren, in Verbindung, k&ouml;nnen sehr Ferntransporte und wesentlich gr&ouml;&szlig;ere Kr&auml;fte ohne die Notwendigkeit zus&auml;tzlicher Faktoren gelten. Wir zeigen dann in-vitro-, dass die wichtige Microtubule-assoziierte Protein, Tau, regelt die Anzahl der engagierten Kinesin Motoren pro Ladung &uuml;ber die lokale Konzentration auf Mikrotubuli. Diese Funktion des Tau bietet einen zuvor unbeachtet Mechanismus zum Verkehr zu regulieren. Indem Sie die motor Wiederbefestigung senken, betrifft Tau Fracht-Reise-Entfernung, treibende Kraft und Fracht Zerstreuung. Wir zeigen auch, dass verschiedene Isoformen von Tau, in Konzentrationen in den Zellen, &auml;hnlich dramatisch anders Potenz. Diese Ergebnisse liefern ein gut definierter Mechanismus wie ver&auml;nderte Tau Isoform Ebenen Verkehr beeintr&auml;chtigen und dadurch zu Neurodegeneration ohne die Notwendigkeit von jeder anderen Weg f&uuml;hren k&ouml;nnte.

Multiple-Motor basierte Transport und seine Verordnung von Tau.

We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau's regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels--where kinesin cannot bind microtubules (MTs)--dynein can still contact MTs. The difference between tau's effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end-directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform-specific effect is not because of steric interference of tau's projection domains but rather because of tau's interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.

Tuning microtubule-based transport through filamentous MAPs: the problem of dynein.

Wir k&uuml;rzlich vorgeschlagen, dass Regulierung des Einzel-zu-mehreren motor &Uuml;bergangs eine wahrscheinlich Strategie zur Regulierung von Kinesin-basierte Transport in-vivo war. In dieser Studie haben wir ein in-vitro-Perlen-Assay, gepaart mit einer optischen Falle verwenden, um zu untersuchen, wie diese vorgeschlagenen Regulierungsmechanismus Dynein-basierte Transport auswirkt. Wir zeigen, dass Tau der Verordnung Kinesin Funktion fortgesetzt werden kann, ohne zu st&ouml;ren Dynein-basierte Transport. Kontaktieren Sie &uuml;berraschenderweise in extrem hohe Tau k&ouml;nnen Ebenen--wo Kinesin Mikrotubuli (MTs)--Dynein binden kann noch MTs. Der Unterschied zwischen Tau die Auswirkungen auf die Motilit&auml;t Kinesin und Dynein-basierte schl&auml;gt vor, dass Tau verwendet werden kann, um relative Menge des Plus-Ende und unter der Regie von Minus-Ende zu optimieren. Wie im Fall der Muskelkontraktion finden wir, dass die 3RS Isoform von Tau ein potenter Inhibitor der Dynein-Bindung an MTs. Wir zeigen, dass diese Isoform-spezifische Wirkung nicht wegen sterische Einmischung der Tau der Projektion Domains sondern wegen des Tau Interaktionen mit dem Motor an der MT-Oberfl&auml;che. Dennoch, wir beobachten eine bescheidene sterische Einmischung Wirkung von Tau Weg von der MT und diskutieren die m&ouml;glichen Auswirkungen dieser f&uuml;r motor Molek&uuml;lstruktur.

Optimierung auf der Grundlage von Microtubule Transport durch filament&ouml;sen Karten: das Problem der Dynein.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

Nuclear envelope in nuclear positioning and cell migration.

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.

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

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.

Article	Year
Multiple-motor based transport and its regulation by Tau. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 17190808	2007
Tuning microtubule-based transport through filamentous MAPs: the problem of dynein. Traffic (Copenhagen, Denmark)\| PubMed ID: 18373727	2008
Bringing KASH under the SUN: the many faces of nucleo-cytoskeletal connections. The Journal of cell biology\| PubMed ID: 19687252	2009
UnLINCing the nuclear envelope: towards an understanding of the physiological significance of nuclear positioning. Biochemical Society transactions\| PubMed ID: 22103527	2011
Analyses of dynein heavy chain mutations reveal complex interactions between dynein motor domains and cellular dynein functions. Genetics\| PubMed ID: 22649085	2012
The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration. Scientific reports\| PubMed ID: 22761994	2012
BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures. Molecular biology of the cell\| PubMed ID: 22956769	2012
LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina. PloS one\| PubMed ID: 23071752	2012
Developmental regulation of linkers of the nucleoskeleton to the cytoskeleton during mouse postnatal retinogenesis. Nucleus (Austin, Tex.)\| PubMed ID: 23974729	2013
Temporal and tissue-specific disruption of LINC complexes in vivo. Genesis (New York, N.Y. : 2000)\| PubMed ID: 24550182	2014
Nuclear envelope in nuclear positioning and cell migration. Advances in experimental medicine and biology\| PubMed ID: 24563361	2014
A variant of Nesprin1 giant devoid of KASH domain underlies the molecular etiology of autosomal recessive cerebellar ataxia type I. Neurobiology of disease\| PubMed ID: 25843669	2015
Nuclear envelope: positioning nuclei and organizing synapses. Current opinion in cell biology\| PubMed ID: 26079712	2015

David Razafsky

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