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1.  PI(4,5)P2-dependent microdomain assemblies capture microtubules to promote and control leading edge motility 
The Journal of Cell Biology  2005;169(1):151-165.
The lipid second messenger PI(4,5)P2 modulates actin dynamics, and its local accumulation at plasmalemmal microdomains (rafts) might mediate regulation of protrusive motility. However, how PI(4,5)P2-rich rafts regulate surface motility is not well understood. Here, we show that upon signals promoting cell surface motility, PI(4,5)P2 directs the assembly of dynamic raft-rich plasmalemmal patches, which promote and sustain protrusive motility. The accumulation of PI(4,5)P2 at rafts, together with Cdc42, promotes patch assembly through N-WASP. The patches exhibit locally regulated PI(4,5)P2 turnover and reduced diffusion-mediated exchange with their environment. Patches capture microtubules (MTs) through patch IQGAP1, to stabilize MTs at the leading edge. Captured MTs in turn deliver PKA to patches to promote patch clustering through further PI(4,5)P2 accumulation in response to cAMP. Patch clustering restricts, spatially confines, and polarizes protrusive motility. Thus, PI(4,5)P2-dependent raft-rich patches enhance local signaling for motility, and their assembly into clusters is regulated through captured MTs and PKA, coupling local regulation of motility to cell polarity, and organization.
PMCID: PMC2171909  PMID: 15809307
2.  A role for cofilin and LIM kinase in Listeria-induced phagocytosis 
The Journal of Cell Biology  2001;155(1):101-112.
The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1−, or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.
PMCID: PMC2150789  PMID: 11571311
Listeria monocytogenes; cytoskeleton; Met; InlB; actin-based motility
3.  Alterations at the Intercalated Disk Associated with the Absence of Muscle Lim Protein 
The Journal of Cell Biology  2001;153(4):763-772.
In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain– containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.
PMCID: PMC2192386  PMID: 11352937
dilated cardiomyopathy; N-RAP; tropomodulin; adherens junction; gap junction
4.  Shared and Unique Roles of Cap23 and Gap43 in Actin Regulation, Neurite Outgrowth, and Anatomical Plasticity 
The Journal of Cell Biology  2000;149(7):1443-1454.
CAP23 is a major cortical cytoskeleton–associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.
PMCID: PMC2175140  PMID: 10871284
neurite outgrowth; neuromuscular junction; synaptic plasticity; actin dynamics; nerve sprouting
5.  Gap43, Marcks, and Cap23 Modulate Pi(4,5p)2 at Plasmalemmal Rafts, and Regulate Cell Cortex Actin Dynamics through a Common Mechanism 
The Journal of Cell Biology  2000;149(7):1455-1472.
The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P2, and promote its retention and clustering. Binding and modulation of PI(4,5)P2 depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P2 modulation. In the neuronlike cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by ΔED mutants. Agents that globally mask PI(4,5)P2 mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(ΔED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P2 modulating proteins, upstream of actin and cell cortex dynamics regulation.
PMCID: PMC2175130  PMID: 10871285
neurite outgrowth; cell spreading; anatomical plasticity; actin recruitment; lipid microdomain
6.  Accumulation of Muscle Ankyrin Repeat Protein Transcript Reveals Local Activation of Primary Myotube Endcompartments during Muscle Morphogenesis  
The Journal of Cell Biology  1997;139(5):1231-1242.
The characteristic shapes and positions of each individual body muscle are established during the process of muscle morphogenesis in response to patterning information from the surrounding mesenchyme. Throughout muscle morphogenesis, primary myotubes are arranged in small parallel bundles, each myotube spanning the forming muscles from end to end. This unique arrangement potentially assigns a crucial role to primary myotube end regions for muscle morphogenesis.
We have cloned muscle ankyrin repeat protein (MARP) as a gene induced in adult rat skeletal muscle by denervation. MARP is the rodent homologue of human C-193 (Chu, W., D.K. Burns, R.A. Swerick, and D.H. Presky. 1995. J. Biol. Chem. 270:10236–10245) and is identical to rat cardiac ankyrin repeat protein. (Zou, Y., S. Evans, J. Chen, H.-C. Kuo, R.P. Harvey, and K.R. Chien. 1997. Development. 124:793–804). In denervated muscle fibers, MARP transcript accumulated in a unique perisynaptic pattern. MARP was also expressed in large blood vessels and in cardiac muscle, where it was further induced by cardiac hypertrophy. During embryonic development, MARP was expressed in forming skeletal muscle. In situ hybridization analysis in mouse embryos revealed that MARP transcript exclusively accumulates at the end regions of primary myotubes during muscle morphogenesis. This closely coincided with the expression of thrombospondin-4 in adjacent prospective tendon mesenchyme, suggesting that these two compartments may constitute a functional unit involved in muscle morphogenesis. Transfection experiments established that MARP protein accumulates in the nucleus and that the levels of both MARP mRNA and protein are controlled by rapid degradation mechanisms characteristic of regulatory early response genes. The results establish the existence of novel regulatory muscle fiber subcompartments associated with muscle morphogenesis and denervation and suggest that MARP may be a crucial nuclear cofactor in local signaling pathways from prospective tendon mesenchyme to forming muscle and from activated muscle interstitial cells to denervated muscle fibers.
PMCID: PMC2140219  PMID: 9382869
7.  Intrinsic Neuronal Determinants Locally Regulate Extrasynaptic and Synaptic Growth at the Adult Neuromuscular Junction 
The Journal of Cell Biology  1997;136(3):679-692.
Long-term functional plasticity in the nervous system can involve structural changes in terminal arborization and synaptic connections. To determine whether the differential expression of intrinsic neuronal determinants affects structural plasticity, we produced and analyzed transgenic mice overexpressing the cytosolic proteins cortical cytoskeleton–associated protein 23 (CAP-23) and growth-associated protein 43 (GAP-43) in adult neurons.
Like GAP-43, CAP-23 was downregulated in mouse motor nerves and neuromuscular junctions during the second postnatal week and reexpressed during regeneration. In transgenic mice, the expression of either protein in adult motoneurons induced spontaneous and greatly potentiated stimulus-induced nerve sprouting at the neuromuscular junction. This sprouting had transgene-specific features, with CAP-23 inducing longer, but less numerous sprouts than GAP-43. Crossing of the transgenic mice led to dramatic potentiation of the sprout-inducing activities of GAP-43 and CAP-23, indicating that these related proteins have complementary and synergistic activities. In addition to ultraterminal sprouting, substantial growth of synaptic structures was induced. Experiments with pre- and postsynaptic toxins revealed that in the presence of GAP-43 or CAP-23, sprouting was stimulated by a mechanism that responds to reduced transmitter release and may be independent of postsynaptic activation.
These results demonstrate the importance of intrinsic determinants in structural plasticity and provide an experimental approach to study its role in nervous system function.
PMCID: PMC2134288  PMID: 9024697
8.  Diverse Modes of Axon Elaboration in the Developing Neocortex 
PLoS Biology  2005;3(8):e272.
The development of axonal arbors is a critical step in the establishment of precise neural circuits, but relatively little is known about the mechanisms of axonal elaboration in the neocortex. We used in vivo two-photon time-lapse microscopy to image axons in the neocortex of green fluorescent protein-transgenic mice over the first 3 wk of postnatal development. This period spans the elaboration of thalamocortical (TC) and Cajal-Retzius (CR) axons and cortical synaptogenesis. Layer 1 collaterals of TC and CR axons were imaged repeatedly over time scales ranging from minutes up to days, and their growth and pruning were analyzed. The structure and dynamics of TC and CR axons differed profoundly. Branches of TC axons terminated in small, bulbous growth cones, while CR axon branch tips had large growth cones with numerous long filopodia. TC axons grew rapidly in straight paths, with frequent interstitial branch additions, while CR axons grew more slowly along tortuous paths. For both types of axon, new branches appeared at interstitial sites along the axon shaft and did not involve growth cone splitting. Pruning occurred via retraction of small axon branches (tens of microns, at both CR and TC axons) or degeneration of large portions of the arbor (hundreds of microns, for TC axons only). The balance between growth and retraction favored overall growth, but only by a slight margin. Given the identical layer 1 territory upon which CR and TC axons grow, the differences in their structure and dynamics likely reflect distinct intrinsic growth programs for axons of long projection neurons versus local interneurons.
Long-term in vivo two-photon imaging of developing thalamocortical and cajal-retzius axons reveals distinct modes of axon elaboration in neocortex.
PMCID: PMC1180514  PMID: 16026180

Results 1-8 (8)