Bioactive peptides in the juxtamembrane regions of proteins are involved in many signaling events. The juxtamembrane regions of cadherins were examined for the identification of bioactive regions. Several peptides spanning the cytoplasmic juxtamembrane regions of E- and N-cadherin were synthesized and assessed for the ability to influence TGFβ responses in epithelial cells at the gene expression and protein levels. Peptides from regions closer to the membrane appeared more potent inhibitors of TGFβ signaling, blocking Smad3 phosphorylation. Thus inhibiting nuclear translocation of phosphorylated Smad complexes and subsequent transcriptional activation of TGFβ signal propagating genes. The peptides demonstrated a peptide-specific potential to inhibit other TGFβ superfamily members, such as BMP4.
BMP4; E-cadherin; Jagged; N-cadherin; Smad; TGFβ; adherens junctions; palmitic acid; peptide
While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.
skeletal muscle; aging; progenitor cells; fiber branching; contractility
We recently identified the atypical myosin, Myosin VI, as a component of epithelial cell-cell junctions that interacts with E-cadherin. Recombinant proteins bearing the cargo-binding domain of Myosin VI (Myo VI-CBD) or the cytoplasmic tail of E-cadherin can interact directly with one another. In this report we further investigate the molecular requirements of the interaction between Myo VI-CBD and E-cadherin combining truncation mutation analysis with in vitro binding assays. We report that a short (28 amino acid) juxtamembrane region of the cadherin cytoplasmic tail is sufficient to bind Myo VI-CBD. However, central regions of the cadherin tail adjacent to the juxtamembrane sequence also display binding activity for Myo VI-CBD. It is therefore possible that the cadherin tail bears two binding sites for Myosin VI, or an extended binding site that includes the juxtamembrane region. Nevertheless, our biochemical data highlight the capacity for the juxtamembrane region to interact with functionally-significant cytoplasmic proteins.
E-cadherin; myosin VI; cytoskeleton; protein-protein interaction; Epithelia
Lymphocytes such as T cells, B cells and natural killer (NK) cells form specialized contacts, called immunological synapses, with other cells in order to engage in specific intercellular communication and killing. Synapse formation is associated with the polarization of the microtubule-organizing center (MTOC) toward the contact site, which enables the directional secretion of cytokines and lytic factors. Although MTOC reorientation to the synapse is crucial for lymphocyte function, it has been difficult to study because of technical constraints. We have developed a photoactivation and imaging strategy that enables high-resolution analysis of cytoskeletal dynamics in individual T cells. Using this approach, we have demonstrated that the lipid second messenger diacylglycerol plays a crucial role in promoting MTOC reorientation by recruiting three members of the protein kinase C family to the synapse. Here, I will discuss these results along with studies from other labs, which have explored the role of polarity-inducing protein complexes after synapse formation. I will also propose a two-step model for MTOC reorientation in lymphocytes that reflects what we now know about the subject. Finally, I will consider the extent to which lymphocyte polarity resembles analogous cell polarity systems in other cell types.
polarity; T cell; microtubule; cytoskeleton; signaling; lymphocyte; chemical biology
Skeletal muscle exhibits strikingly regular intracellular sorting of actin and tropomodulin (Tmod) isoforms, which are essential for efficient muscle contraction. A recent study from our laboratory demonstrates that the skeletal muscle sarcoplasmic reticulum (SR) is associated with cytoplasmic γ-actin (γcyto-actin) filaments, which are predominantly capped by Tmod3. When Tmod3 is experimentally induced to vacate its SR compartment, the cytoskeletal organization of SR-associated γcyto-actin is perturbed, leading to SR swelling, depressed SR Ca2+ release and myofibril misalignment. Based on these findings, Tmod3-capped γcyto-actin filaments mechanically stabilize SR structure and regulate SR function via a novel lateral linkage. Furthermore, by placing these findings in the context of studies in nonmuscle cells, we conclude that Tmodcapped actin filaments are emerging as critical regulators of membrane stability and physiology in a broad assortment of cell types.
cell membrane; cytoskeletal connections; muscle contraction; sarcomere; sarcoplasmic reticulum; thin filament
Mechanoelectrical transduction (MET), the conversion of mechanical stimuli into electrical signals operated by the sensory cells of the inner ear, enables hearing and balance perception. Crucial to this process are the tip-links, oblique fibrous filaments that interconnect the actin-filled stereocilia of different rows within the hair bundle, and mechanically gate MET channels. In a recent study, we observed a complete regression of stereocilia from the short and medium but not the tall row upon the disappearance of the tip-links caused by the loss of one of their components, cadherin-23, or of one of their anchoring proteins, sans, in the auditory organs of engineered mutant mice. This indicates the existence of a coupling between the MET and F-actin polymerization machineries at the tips of the short and medium stereocilia rows in cochlear hair bundles. Here, we first present our findings in the mutant mice, and then discuss the possible effects of the tip-link tension on stereocilia F-actin polymerization, acting either directly or through Ca2+-dependent mechanisms that involve the gating of MET channels.
hair cell; hair bundle; stereocilia; mechanoelectrical transduction (MET); tip-link; sans protein; actin polymerization
Tropomyosins are rod-like dimers which form head-to-tail polymers along the length of actin filaments and regulate the access of actin binding proteins to the filaments.1 The diversity of tropomyosin isoforms, over 40 in mammals, and their role in an increasing number of biological processes presents a challenge both to experienced researchers and those new to this field. The increased appreciation that the role of these isoforms expands beyond that of simply stabilizing actin filaments has lead to a surge of reagents and techniques to study their function and mechanisms of action. This report is designed to provide a basic guide to the genes and proteins and the availability of reagents which allow effective study of this family of proteins. We highlight the value of combining multiple techniques to better evaluate the function of different tm isoforms and discuss the limitations of selected reagents. Brief background material is included to demystify some of the unfortunate complexity regarding this multi-gene family of proteins including the unconventional nomenclature of the isoforms and the evolutionary relationships of isoforms between species. Additionally, we present step-by-step detailed experimental protocols used in our laboratory to assist new comers to the field and experts alike.
tropomyosin; isoforms; cytoskeleton; reagents; antibodies; multi-gene family
Aspects of cellular architecture, such as cytoskeletal asymmetry cues, play critical roles in directing the placement of organelles and establishing the sites of their formation. In the model green alga Chlamydomonas, the photosensory eyespot occupies a defined position in relation to the flagella and microtubule cytoskeleton. Investigations into the cellular mechanisms of eyespot placement and assembly have aided our understanding of the interplay between cytoskeletal and plastid components of the cell. The eyespot, which must be assembled anew after each cell division, is a multi-layered organelle consisting of stacks of carotenoid-filled pigment granules in the chloroplast and rhodopsin photoreceptors in the plasma membrane. Placement of the eyespot is determined on both the latitudinal and longitudinal axes of the cell by the daughter four-membered (D4) microtubule rootlet. Recent findings have contributed to the hypothesis that the eyespot photoreceptor molecules are directed from the Golgi to the daughter hemisphere of the cell and trafficked along the D4 microtubule rootlet. EYE2, a chloroplast-envelope protein, forms an elliptical patch together with the photoreceptors and establishes the site for assembly of the pigment granule arrays in the chloroplast, connecting the positioning information of the cytoskeleton to assembly of the pigment granule arrays in the chloroplast.
Chlamydomonas; eyespot; organelle placement; organelle assembly; microtubule rootlet; asymmetry; photoreception
Metastasis involves tumor cells moving through tissues and crossing tissue boundaries, which requires cell migration, remodeling of cell-to-cell contacts and interactions with the extracellular matrix. Individual tumor cells move in three-dimensional environments with either a rounded “ameboid” or an elongated “mesenchymal” morphology. These two modes of movement are tightly regulated by Rho family GTPases: elongated movement requires activation of Rac1, whereas rounded movement engages specific Cdc42 and Rho signaling pathways. It has been known for some time that events unfolding downstream of Ras GTPases are also involved in regulating multiple aspects of cell migration and invasion. More recently, RasGRF2—a Ras activator—has been identified as a suppressor of rounded movement, by inhibiting the activation of Cdc42, independently of its capacity to activate Ras. Here, we discuss how Rho and Ras signals can either cooperate or oppose each other in the regulation of cell migration and invasion.
Ras GTPases; Rho GTPases; RasGRF; cell migration and invasion; metastasis
The actin cytoskeleton plays essential roles in cell polarization and cell morphogenesis of the budding yeast Saccharomyces cerevisiae. Yeast cells utilize formin-generated actin cables as tracks for polarized transport, which forms the basis for a positive feedback loop driving Cdc42-dependent cell polarization. Previous studies on cable organization mostly focused on polarized actin cables in budded cells and their role as relatively static tracks for myosin-dependent organelle transport. Using quantitative live cell imaging, we have recently characterized the dynamics of cortical actin cables throughout the yeast cell cycle. Surprisingly, randomly oriented actin cables in G1 cells exhibited the highest level of dynamics, while cable dynamics was markedly slowed down upon cell polarization. We further demonstrated that the rapid dynamics of randomly oriented cables were driven by the formin Bni1 and Myosin V. Our data suggested a precise spatio-temporal regulation of the two yeast formins, as well as an unexpected mechanism of actin cable rearrangement through myosins. Here we discuss the immediate significance of these findings, which illustrates the importance of generating randomness for cellular organization.
actin; formin; myosin; polarity; self organization
Adherens junctions (AJs) and tight junctions (TJs) represent key adhesive structures that regulate the apico-basal polarity and barrier properties of epithelial layers. AJs and TJs readily undergo disassembly and reassembly during normal tissue remodeling and disruption of epithelial barriers in diseases. Such junctional plasticity depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, however the interplay between these cellular structures remains poorly understood. Recent studies highlighted the spectrin-adducin-based membrane skeleton as an emerging regulator of AJ and TJ integrity and remodeling. Here we discuss new evidences implicating adducin, spectrin and other membrane skeleton proteins in stabilization of epithelial junctions and regulation of junctional dynamics. Based on the known ability of the membrane skeleton to link cortical actin filaments to the plasma membrane, we hypothesize that the spectrin-adducin network serves as a critical signal and force transducer from the actomyosin cytoskeleton to junctions during remodeling of AJs and TJs.
adherens junctions; tight junctions; permeability; membrane skeleton; actomyosin; contractility; calcium switch
The function of nuclear actin is poorly understood. It is known to be a discrete component of several chromatin-modifying complexes. Nevertheless, filamentous forms of actin are important for various nuclear processes as well. Nuclear actin is often associated with nuclear actin-related protein Arp4 and other actin-related proteins like Arp8 in the INO80 chromatin remodeler. We recently determined the crystal structure of S. cerevisiae Arp4 that explains why Arp4 is unable to form actin like filaments and shows that it is constitutively bound to an ATP nucleotide. More interestingly, in vitro activities of Arp4 and Arp8 seem to be directed towards stabilizing monomeric actin and to integrate it stoichiometrically into the INO80 complex. Based on this activity, we discuss possible roles of nuclear Arps in chromatin modifying complexes and in regulating more general aspects of nuclear actin dynamics.
actin-related proteins; chromatin remodeling; INO80 complex; nuclear actin
Polar residues lying between adjacent α-helical chains of coiled-coils often contribute to coiled-coil curvature and flexibility, while more typical core hydrophobic residues anneal the chains together. In tropomyosins, ranging from smooth and skeletal muscle to cytoplasmic isoforms, a highly conserved Asp at residue 137 places negative charges within the tropomyosin coiled-coil core in a position which may affect the conformation needed for tropomyosin binding and regulatory movements on actin. Proteolytic susceptibility suggested that substituting a canonical Leu for the naturally occurring Asp at residue 137 increases inter-chain rigidity by stabilizing the tropomyosin coiled-coil. Using molecular dynamics, we now directly assess changes in coiled-coil curvature and flexibility caused by such mutants. Although the coiled-coil flexibility is modestly diminished near the residue 137 mutation site, as expected, a delocalized increase in flexibility along the overall coiled-coil is observed. Even though the average shape of the D137L tropomyosin is straighter than that of wild-type tropomyosin, it is still capable of binding actin due to this increase in flexibility. We conclude that the conserved, non-canonical Asp-137 destabilizes the local structure resulting in a local flexible region in the middle of tropomyosin that normally is important for tropomyosin steady-state equilibrium position on actin.
Coiled-coil; Flexibility; Molecular Dynamics; heptad repeat; tropomyosin
After peripheral nerve injuries, Wallerian degeneration starts with a stereotypic fragmentation of myelin sheath into myelin ovoids, which occur near Schmidt-Lantermann incisures (SLI). This demyelination process requires a dramatic change in cytoskeletal structures in Schwann cells. We have recently shown that actin polymerization around SLI is an important step for cleavage of the myelin sheath. We described that Rac1 GTP ase regulates actin polymerization in SLI after injury. It has been previously reported that Rac-dependent cytoskeletal reorganization also plays an important role in myelination during the development of peripheral nerves. Thus, our findings suggest that Rac-dependent actin polymerization controls both myelination and demyelination in the peripheral nerves. We further discuss our new findings in relation to Schwann cell dedifferentiation and segmental demyelination.
Schwann cells; wallerian degeneration; myelin fragmentation; actin polymerization; Rac GTPases; Schmidt-Lantermann incisures
We have previously reported that actin undergoes a conformational transition (which we named “maturation”) during polymerization, and that the actin-binding protein, caldesmon (CaD), when added at an early phase of polymerization, interferes with this process (Huang et al. J Biol Chem 2010; 285:71). The pre-transition filament is characterized by relatively low pyrene-fluorescence intensity when pyrene-labeled actin is used as a reporter of subunit assembly into filaments, whereas the mature filament emits a characteristic enhanced fluorescence. Previously reported co-sedimentation experiments suggest that filament formation is not inhibited by the presence of CaD, despite blocking the transition associated with filament maturation. In this study we visualized structural effects of CaD on the assembly of actin filaments by TIRF and electron microscopy. CaD-free actin forms “rough” filaments with irregular edges and indistinct subunit organization during the initial phase (∼20 min under our conditions) of polymerization as reported previously by others (Steinmetz et al. J Cell Biol 1997; 138:559; Galinska-Rakoczy et al. J Mol Biol 2009; 387:869), which most likely correspond to the pre-transition state preceding the maturation step. Later during the polymerization process “mature” filaments exhibit a smoother F-actin appearance with easily detectible double helically arranged actin subunits. While the inclusion of the actin-binding domain of CaD during actin polymerization does not affect the elongation rate, it is associated with a prolonged pre-transition phase, characterized by a delayed alteration (rough to smooth) of the appearance of filaments, consistent with a later onset of the maturation process.
actin; polymerization; caldesmon; TIRF microscopy; electron microscopy; conformational change; cytoskeleton dynamics
Transforming acidic coiled coil 3 (TACC3) is a non-motor microtubule-associated protein (MAP) that is important for mitotic spindle stability and organization. The exact mechanism by which TACC3 acts at microtubules to stabilize the spindle has been unclear. However, several recent studies identified that the TACC3 complex at microtubules contains clathrin in addition to its previously identified binding partner, colonic and hepatic tumor overexpressed gene (ch-TOG). In this complex, phosphorylated TACC3 interacts directly with both ch-TOG and clathrin heavy chain, promoting accumulation of all complex members at the mitotic spindle. This complex stabilizes kinetochore fibers within the spindle by forming cross-bridges that link adjacent microtubules in these bundles. So, TACC3 is an adaptor that recruits ch-TOG and clathrin to mitotic microtubules, in an Aurora A kinase-regulated manner. In this mini-review we will describe the recent advances in the understanding of TACC 3 function and present a model that pulls together these new data with previous observations.
TACC3; clathrin; ch-TOG; kinetochore fiber; cross-linking
In many animals, germ-cell fate is specified by inheritance of the germ plasm, which is enriched in maternal RNAs and proteins. Assembly of the Drosophila germ (pole) plasm begins with the localization and translation of oskar (osk) RNA at the oocyte posterior pole. osk RNA produces two isoforms, long and short Osk. Short Osk recruits other pole plasm components, and long Osk restricts them to the oocyte cortex. Although molecular functions of long Osk remain mysterious, it is known to be involved in endocytic activation and actin cytoskeletal remodeling. We identified several vesicular trafficking machinery components that act downstream of long Osk in pole plasm assembly. These included the Rab5 effector protein Rabenosyn-5 (Rbsn-5) and the Golgi/endosomal protein Mon2, both of which were crucial for Osk-induced actin remodeling and the anchoring of pole plasm components. We propose that, in response to long Osk, the Rab5/Rbsn-5-dependent endocytic pathway promotes the formation of specialized vesicles, and Mon2 acts on these vesicles as a scaffold to instruct actin nucleators like Cappuccino and Spire to remodel the actin cytoskeleton, which anchors pole plasm components to the cortex. This mechanism may be applicable to the asymmetric localization of macromolecular structures such as protein-RNA complexes in other systems.
germ plasm; oskar; cell polarization; vesicle trafficking; golgi; endosome; actin cytoskeleton
Cell wounding is a common event in the life of many cell types, and the capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Cell wounding is most frequent in tissues exposed to high levels of stress. Survival of such plasma membrane disruptions requires rapid resealing to prevent the loss of cytosolic components, to block Ca2+ influx and to avoid cell death. In addition to patching the torn membrane, plasma membrane and cortical cytoskeleton remodeling are required to restore cell function. Although a general understanding of the cell wound repair process is in place, the underlying mechanisms of each step of this response are not yet known. We have developed a model to study single cell wound repair using the early Drosophila embryo. Our system combines genetics and live imaging tools, allowing us to dissect in vivo the dynamics of the single cell wound response. We have shown that cell wound repair in Drosophila requires the coordinated activities of plasma membrane and cytoskeleton components. Furthermore, we identified an unexpected role for E-cadherin as a link between the contractile actomyosin ring and the newly formed plasma membrane plug.
wound repair; single cell; plasma membrane; cytoskeleton; actin; myosin; E-cadherin; purse string; Drosophila
Mitotic Centromere Associated Kinesin (MCAK) is a potent microtubule depolymerizing and catastrophe-inducing factor, which uses the energy of ATP hydrolysis to destabilize microtubule ends. MCAK is localized to inner centromeres, kinetochores and spindle poles of mitotic cells, and is also present in the cytoplasm. Both in interphase and in mitosis, MCAK can specifically accumulate at the growing microtubule ends. Here we discuss the mechanisms, which modulate subcellular localization and activity of MCAK through the interaction with the End Binding (EB) proteins and phosphorylation.
microtubule; mitosis; kinesin; kinase; phosphorylation; EB1; MCAK
The centrosome is a major microtubule-organizing center in animal cells, and its intracellular positioning is critical for defining intracellular architecture. The centrosome positions itself at the cell center. Centrosome centration depends on the microtubule cytoskeleton. To accomplish robust centration regardless of the cell size or cell shape, it has been assumed that the force mediated by the microtubules depends on microtubule length. However, a concrete mechanism to generate forces to pull the centrosome in a microtubule length-dependent manner has been elusive. Recently, we successfully demonstrated that centrosome-directed movement of intracellular organelles along microtubules drives centrosome centration in the Caenorhabditis elegans early embryo. Based on this observation, we proposed the centrosome-organelle mutual pulling model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first experiment-based model that accounts for the microtubule length-dependent pulling force generated in the cytoplasm contributing to centrosome centration. Intriguingly, this model is consistent with a recent estimation that the pulling force is proportional to the cubic length of microtubules.
centrosome centration; microtubule length dependency; dynein; centrosome-organelle mutual pulling model
Stable attachment of kinetochores to spindle microtubules is essential for accurate chromosome segregation. We have shown that a kinetochore-associated formin protein, mDia3, contributes to the generation of stable kinetochore-microtubule attachment. The published report reviewed here shows an essential role of mDia3 in achieving metaphase chromosome alignment, and this function is directly regulated by Aurora B phosphorylation. Aurora B is a central component during the capture of spindle microtubules by kinetochores, in which it selectively eliminates incorrect attachments by phosphorylating a group of microtubule binding proteins at kinetochores to reduce their microtubule binding affinity. Here, we discuss the roles of Aurora B kinase and its substrates in achieving proper kinetochore-microtubule attachment.
Aurora B; mDia3; kinetochore; microtubule; mitosis
Striated muscle cells display an extremely regular assembly of their actin cytoskeleton that contributes to the contractile elements, the myofibrils. How this assembly is initiated and how these structures are maintained is still unclear. We have recently shown that striated muscle expresses a specific isoform of the formin protein family member FHOD3, which is characterised by the presence of a CK2 phosphorylation site at the C-terminal end of the formin homology domain 2 (FH2). Phosphorylated muscle FHOD3 displays a different subcellular localisation, namely to the myofibrils, and also has increased stability compared to un-phosphorylated or non muscle FHOD3. In addition, we could show that muscle FHOD3 is involved in myofibril maintenance in cultured cardiomyocytes and that its presence dramatically enhances the reconstitution of cardiac actin filaments after depolymerisation. Since FHOD3 expression levels and in particular that of the muscle isoform are also decreased in different types of cardiomyopathy, we postulate a crucial role for this protein in the maintenance of a fully functional cardiac cytoarchitecture.
heart; development; actin filament; formin; sarcomere
Tardigrades, also known as water bears, have somatic muscle fibers that are responsible for movement of their body and legs. These muscle fibers contain thin and thick filaments in a non-striated pattern. However, the regulatory mechanism of muscle contraction in tardigrades is unknown. In the absence of extensive molecular and genomic information, we detected a protein of 31 kDa in whole lysates of tardigrades that cross-reacted with the antibody raised against nematode troponin I (TnI). TnI is a component of the troponin complex that regulates actin-myosin interaction in a Ca2+-dependent and actin-linked manner. This TnI-like protein was co-extracted with actin in a buffer containing ATP and EGTA, which is known to induce relaxation of a troponin-regulated contractile system. The TnI-like protein was specifically expressed in the somatic muscle fibers in adult animals and partially co-localized with actin filaments in a non-striated manner. Interestingly, the pharyngeal muscle did not express this protein. These observations suggest that the non-striated somatic muscle of tardigrades has an actin-linked and troponin-regulated system for muscle contraction.
troponin; actin; actin-linked regulation; non-striated muscle; tardigrades
During mitosis, the Golgi complex undergoes a multi-step fragmentation process that is instrumental to its correct partitioning into the daughter cells. To prepare for this segregation, the Golgi ribbon is initially separated into individual stacks during the G2 phase of the cell cycle. Then, at the onset of mitosis, these individual stacks are further disassembled into dispersed fragments. Inhibition of this Golgi fragmentation step results in a block or delay of G2/M transition, depending on the experimental approach. Thus, correct segregation of the Golgi complex appears to be monitored by a ‘Golgi mitotic checkpoint’. Using a microinjection-based approach, we recently identified the first target of the Golgi checkpoint, whereby a block of this Golgi fragmentation impairs recruitment of the mitotic kinase Aurora-A to, and its activation at, the centrosomes. Overexpression of Aurora-A can override this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. We have also shown that this block of Aurora-A recruitment to the centrosomes is not mediated by the known mechanisms of regulation of Aurora-A function. Here we discuss our findings in relation to the known functions of Aurora-A.
mitotic checkpoint; golgi complex; cell cycle; G2; aurora-A; cancer
PSD-95, a synaptic scaffolding protein, plays important roles in the regulation of dendritic spine morphology and glutamate receptor signaling. We have recently shown that PSD-95 also plays an extrasynaptic role during development. PSD-95 shapes dendrite branching patterns in cultured rat hippocampal neurons by altering microtubule dynamics via an association with the microtubule end-binding protein-3 (EB3). We discovered that PSD-95 interacts directly with EB3 and that the result of this interaction decreases EB3 binding to and EB3 comet lifetime on microtubules. This decrease in lifetime also correlates to decreased dendrite branching. Here we present an additional effect of PSD-95 overexpression on microtubules. Neurons that overexpress PSD-95 show increased distance between microtubules in a manner that is not fully dependent on the interaction between PSD-95 and EB3. We discuss these new data in the context of the role of PSD-95 in shaping the dendritic arbor, and we extend our findings to include a discussion of how PSD-95 may guide neurons toward a more mature and synapse-oriented growth stage.
PSD-95; EB3; dendrites; microtubule; spines; APC; neuron