• Molecules and Cells
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• 제37권4호
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• pp.307-313
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• 2014

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. Rap-GAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of migrating cells. Here, we examined localization of truncated RapGAP3 proteins and found that the I/LWEQ domain in the central region of RapGAP3 was sufficient for posterior localization in migrating cells, as opposed to leading-edge localization of full-length Rap-GAP3. All truncated proteins accumulated at the leading edge of migrating cells exhibited clear translocation to the cell cortex in response to stimulation, whereas proteins localized to the posterior in migrating cells displayed no translocation to the cortex. The I/LWEQ domain appears to passively accumulate at the posterior region in migrating cells due to exclusion from the extended front region in response to chemoattractant stimulation rather than actively being localized to the back of cells. Our results suggest that posterior localization of the I/LWEQ domain of RapGAP3 is likely related to F-actin, which has probably different properties compared to newly formed F-actin at the leading edge of migrating cells, at the lateral and posterior regions of the cell.

• Molecules and Cells
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• 제24권1호
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• pp.76-82
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• 2007

By combining in situ hybridization (ISH) and immunocytochemistry (IC), microscopic topological localization of mRNAs and proteins can be determined. Although this technique can be applied to a variety of tissues, it is particularly important for use on neuronal cells which are morphologically complex and in which specific mRNAs and proteins are located in distinct subcellular domains such as dendrites and dendritic spines. One common technical problem for combined ISH and IC is that the signal for immunocytochemical localization of proteins often becomes much weaker after conducting ISH. In this manuscript, we report a simplified but robust protocol that allows immunocytochemical localization of proteins after ISH. In this protocol, we fix cultured cortical or hippocampal neurons with 4% paraformaldehyde (PFA), rinse briefly in PBS, and then further fix the cells with $-20^{\circ}C$ methanol. Our method has several major advantages over previously described ones in that (1) it is simple, as it is just consecutive routine fixation procedures, (2) it does not require any special alteration to the fixation procedures such as changes in salt concentration, and (3) it can be used with antibodies that are compatible with either methanol (MeOH-) or PFA-fixed target proteins. To our best knowledge, we are the first to employ this fixation method for fluorescence ISH + IC.

• Molecules and Cells
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• 제21권1호
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• pp.34-41
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• 2006

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.237-242
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• 2005

Predicting the cellular location of an unknown protein gives a valuable information for inferring the possible function of the protein. For more accurate prediction system, we need a good feature extraction method that transforms the raw sequence data into the numerical feature vector, minimizing information loss. In this paper, we propose new methods of extracting underlying features only from the sequence data by computing pairwise sequence alignment scores. In addition, we use composition based features to improve prediction accuracy. To construct an SVM ensemble from separately trained SVM classifiers, we propose specificity based weighted majority voting. The overall prediction accuracy evaluated by the 5-fold cross-validation reached 88.53% for the eukaryotic animal data set. By comparing the prediction accuracy of various feature extraction methods, we could get the biological insight on the location of targeting information. Our numerical experiments confirm that our new feature extraction methods are very useful for predicting subcellular localization of proteins.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.606-611
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• 2000

Dimorphic yeast Candida albicans reversibly switches between the form of yeast and hyphae depending on external conditions. We investigated possible roles of the myosin family in the growth and dimorphic switches of C. albicans with a general myosin ATPase inhibitor, 2,3-butanedione-2-monoxime (BDM). Transition to hyphae as well as proliferation by budding was completely inhibited by BDM at 16 mM. Presence of 16 mM BDM did not affect hyphae-to-bud transition but it blocked budding. The effects of BDM on yeast growth and dimorphic switches were reversible. More than 70% of the BDM-treated cells demonstrated defects in the amount and the polarized localization of F-actin as well as in the shape and migration of the nucleus, suggesting that myosin activities are needed in these cellular processes of C. albicans.

• BMB Reports
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• 제46권2호
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• pp.65-72
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• 2013

In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demon-strating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells.

• Animal cells and systems
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• 제5권3호
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• pp.225-229
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• 2001

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand cellular processes in which Nek2 participates during mammalian development, we investigated the expression and subcellular localization of Nek2 in vivo. The Nek2 protein was detected in spermatocytes and in a fraction of actively dividing ovarian follicle cells and of embryonic tissues. We also observed that Nek2 was localized in both the nucleus and centrosome in embryonic cells. Such localization pattern supports the proposal that Nek2 is a mitotic regulator that is involved in multiple cell cycle events during mammalian development.

• 생명과학회지
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• 제27권11호
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• pp.1369-1375
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• 2017

본 연구의 목적은 넙치 인지질가수분해효소(PLC-${\delta}1$) 3가지 타입의 세포내 특성을 규명하고자 하였다. 일반적으로 인지질가수분해효소(PLC)의 신호전달경로는 핵, 세포막, 세포질에 분포한다고 알려져 있으나, 핵내 위치 메커니즘은 여전히 불분명하다. PoPLC-${\delta}1A$, PoPLC-${\delta}1B$ (Sf)과 PoPLC-${\delta}1B$ (Lf)의 3타입의 유전자들은 각각 핵위치 신호(NLS)와 핵방출서열(NES)을 포함하고 있다. 본 연구에서는, 넙치 3가지 타입 인지질가수분해효소(PLC-${\delta}1$)의 세포내 위치이동 메커니즘 분석을 위해 GFP 벡터에 유전자를 삽입하여 ionomycin과 thasogargin처리 후 세포위치와 이동양상을 공초점 레이저 주사현미경으로 관찰하였다. PoPLC-${\delta}1A$는 PoPLC-${\delta}1B$ (Lf)와 PoPLC-${\delta}1B$ (Sf)가 원형질막에 국한되어 분포할때 세포질과 세포막보다 세포 소기관에 분포되어 있었다. PoPLC-${\delta}1B$ (Lf) 및 PoPLC-${\delta}1$ (Sf)이 핵 세포질내 이동양상을 보이지 않을 때, PoPLC-${\delta}1A$은 ionomycin과 thapsigargin 처리에 의해 핵 내에 축적되는 양상을 나타냈다. 이런 결과는 손상되지 않은 기능적 NES 서열을 포함하는 PoPLC-${\delta}1A$가 어류에서 핵 세포질 내 왕복 및 이동의 주된 역할을 한다는 것을 보여주고 있다.

• 분석과학
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• 제29권1호
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• pp.10-18
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• 2016

Phosphodiesterase(PDE)는 세포내에서 cAMP를 분해하는 효소로 세포의 신호 전달에 중요한 기능을 수행하는 것으로 알려져 왔다. 이전의 연구를 통해 군소에서 클로닝된 PDE4 long-form의 N-말단에 위치하는 16개 아미노산만으로 충분히 원형질막에 타기팅됨을 알 수 있었다. 본 연구에서는 ApPDE4의 N-말단 16개(L(N16))를 주형으로 해서 9-11번째와 15번째 아미노산들(RHW-C)을 무작위적으로 아마노산에 돌연변이를 주어서 세포내 타기팅에 미치는 영향을 분석해 보았다. 본 연구를 통해 원형질막과 골지체로 타기팅되는 돌연변이체들과 골지체로만 타기팅되는 돌연변이체들과, 소포체와 골지체로 동시에 타기팅되는 돌연변이체들과, 세포질에만 위치하는 돌연변이체들을 얻을 수 있었다. 또한, 이러한 타기팅에 palmitoylation이 영향을 주는지 확인하기 위해 palmitoylation 억제제인 2-BR을 처리해보니 대부분의 돌연변이체에서 원형질막 타기팅이 사라지는 것을 확인하였다. 이를통해 palmitoylation이 ApPDE4 돌연변이체들의 원형질막 타기팅에 중요하다는 사실을 확인할 수 있었다. 또한, 이들 돌연변이체들중 골지체로만 타기팅되는 L(N16,C3S/VV/G)-mRFP와 L(N16,C3S/LFS/R)-mRFP와 L(N16,EPL/R)-mRFP들의 경우는 골지체 타기팅에 인지질 중에 하나인 PI4P가 중요한 역할을 하는 것을 알 수 있었다.

• BMB Reports
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• 제43권5호
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• pp.344-348
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• 2010

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Staufen2 is an mRNA-binding protein expressed in the cell bodies and cellular processes of different brain cells. It is notably involved in the transport of dendritic mRNAs along microtubules. Its knockdown expression was shown to change spine morphology and impair synaptic functions. However, the identity of Staufen2-bound mRNAs in brain cells is still completely unknown. As a mean to identify these mRNAs, we immunoprecipitated Staufen2-containing mRNPs from embryonic rat brains and used a genome wide approach to identify Staufen2-associated mRNAs. The genome wide approach identified 1780 mRNAs in Staufen2-containing mRNPs that code for proteins involved in cellular processes such as post-translational protein modifications, RNA metabolism, intracellular transport and translation. These results represent an additional and important step in the characterization of Staufen2- mediated neuronal functions in rat brains.