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1.
Brain Cell Biol ; 35(1): 39-56, 2006 Feb.
Article in English | MEDLINE | ID: mdl-17940912

ABSTRACT

A key event in neurite initiation is the accumulation of microtubule bundles at the neuron periphery. We hypothesized that such bundled microtubules may generate a force at the plasma membrane that facilitates neurite initiation. To test this idea we observed the behavior of microtubule bundles that were induced by the microtubule-associated protein MAP2c. Endogenous MAP2c contributes to neurite initiation in primary neurons, and exogeneous MAP2c is sufficient to induce neurites in Neuro-2a cells. We performed nocodazol washout experiments in primary neurons, Neuro-2a cells and COS-7 cells to investigate the underlying mechanism. During nocodazol washout, small microtubule bundles formed rapidly in the cytoplasm and immediately began to move toward the cell periphery in a unidirectional manner. In neurons and Neuro-2a cells, neurite-like processes extended within minutes and concurrently accumulated bundles of repolymerized microtubules. Speckle microscopy in COS-7 cells indicated that bundle movement was due to transport, not treadmilling. At the periphery bundles remained under a unidirectional force and induced local cell protrusions that were further enhanced by suppression of Rho kinase activity. Surprisingly, this bundle motility was independent of classical actin- or microtubule-based tracks. It was, however, reversed by function-blocking antibodies against dynein. Suppression of dynein expression in primary neurons by RNA interference severely inhibited the generation of new neurites, but not the elongation of existing neurites formed prior to dynein knockdown. Together, these cell biological data suggest that neuronal microtubule-associated proteins induce microtubule bundles that are pushed outward by dynein and locally override inward contraction to initiate neurite-like cell protrusions. A similar force-generating mechanism might participate in spontaneous initiation of neurites in developing neurons.


Subject(s)
Dyneins/metabolism , Microtubules/metabolism , Neurites/physiology , Neurons/physiology , Neurons/ultrastructure , Actins/metabolism , Animals , Antineoplastic Agents/pharmacology , Biological Transport/physiology , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cytoplasm/metabolism , Hippocampus/cytology , Mice , Microtubule-Associated Proteins/metabolism , Microtubules/drug effects , Neuroblastoma , Nocodazole/pharmacology , Polymers/metabolism
2.
Am J Physiol Renal Physiol ; 284(4): F727-36, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12488247

ABSTRACT

Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.


Subject(s)
Kidney/metabolism , Symporters/chemistry , Symporters/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Female , Flounder , Hydrogen-Ion Concentration , Immunohistochemistry , Membrane Potentials/physiology , Mice , Microinjections , Molecular Sequence Data , Oocytes/metabolism , Organ Specificity , Patch-Clamp Techniques , Phosphates/chemistry , Rats , Sequence Alignment , Sodium/chemistry , Sodium-Phosphate Cotransporter Proteins , Sodium-Phosphate Cotransporter Proteins, Type II , Sodium-Phosphate Cotransporter Proteins, Type IIb , Symporters/biosynthesis , Xenopus laevis , Zebrafish , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/chemistry , Zebrafish Proteins/genetics
3.
J Physiol ; 520 Pt 1: 79-89, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10517802

ABSTRACT

1. We report the molecular identification of a Na+-Pi (inorganic phosphate) cotransport system of the NaPi-II protein family from zebrafish intestine. Following a PCR-related strategy, a DNA fragment from intestine-derived RNA was isolated. Rapid amplification of cDNA ends (3'- and 5'-RACE) resulted in the complete sequence (2607 bp) containing an open reading frame of 1893 bp. 2. The NaPi-II-related protein was expressed in Xenopus laevis oocytes and the resulting transport activity was analysed by electrophysiological means. The apparent Km for Pi was 250 microM (96 mM Na+, -60 mV), and voltage-dependent binding of Na+ exhibited a Km of 67.1 mM (1 mM Pi, -60 mV). 3. Interestingly, the overall transport activity was almost insensitive to changes in the holding potential. The apparent affinity for Na+ decreased under hyperpolarizing conditions, whereas Pi binding showed no voltage dependence. Transport activity was inhibited at low pH, which is characteristic for renal NaPi-II isoforms. 4. The expression of the NaPi-II-related isoform was addressed by reverse-transcription PCR. The mRNA could be detected in intestine, liver, eye and kidney. Unexpectedly, a second NaPi-II-related isoform was identified and found to be expressed in kidney, intestine, liver, brain, eye and prominently in testis. In addition, a shorter amplicon was demonstrated to be an antisense transcript related to the NaPi-II intestinal isoform.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Symporters , Zebrafish/physiology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Electrophysiology , Hydrogen-Ion Concentration , Isomerism , Kinetics , Molecular Sequence Data , Oocytes/metabolism , Patch-Clamp Techniques , Polymerase Chain Reaction , Protein Conformation , RNA, Antisense/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Sodium-Phosphate Cotransporter Proteins , Sodium-Phosphate Cotransporter Proteins, Type IIb , Tissue Distribution , Transcription, Genetic/genetics , Xenopus laevis , Zebrafish Proteins
4.
J Exp Biol ; 201(Pt 23): 3135-42, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9808829

ABSTRACT

In vertebrates, the level of inorganic phosphate (Pi) is tightly balanced both inside the cell and in the whole organism. A number of different Na+-dependent Pi cotransport systems involved in Pi homeostasis have been identified and characterized at the molecular level in the past 7 years. The transporters constitute three different protein families denoted NaPi-I, NaPi-II and NaPi-III. NaPi-I from the rabbit was the first member of this family to be cloned. However, it still resists efforts to unravel its physiological role and a clear-cut functional identity: is it a Cl- channel, a Na+/Pi cotransporter, a regulator, or does it perform a combination of these functions? These questions provide a slight taste of the problems associated with orphan genes derived from sequencing projects. The members of the NaPi-II protein family are crucially involved in tightly controlled renal Pi excretion and, as recently discovered, intestinal Pi absorption. The expression and the cellular distribution of NaPi-II in the proximal tubular epithelium are affected by hormonal and metabolic factors known to influence extracellular fluid Pi homeostasis. Recently, the expression of NaPi-II has been demonstrated in osteoclasts and brain; however, the physiological roles of NaPi-II in these tissues remain to be established. The members of the third protein family, NaPi-III, have been identified on the basis of their function as viral receptors. The widespread expression of this family suggests that NaPi-III is involved in supplying the basic cellular metabolic needs for Pi.


Subject(s)
Carrier Proteins/physiology , Multigene Family , Protein Isoforms/physiology , Symporters , Animals , Carrier Proteins/classification , Carrier Proteins/genetics , Cloning, Molecular , Cricetinae , Energy Metabolism , Evolution, Molecular , Fishes/metabolism , Humans , Kidney/metabolism , Mammals/metabolism , Mice , Organ Specificity , Phylogeny , Protein Conformation , Protein Isoforms/genetics , Rabbits , Rats , Sodium-Phosphate Cotransporter Proteins , Stress, Physiological/metabolism
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