1,180 research outputs found
Connecting the Cytoskeleton to the Endoplasmic Reticulum and Golgi
A tendency in cell biology is to divide and conquer. For example, decades of painstaking work have led to an understanding of endoplasmic reticulum (ER) and Golgi structure, dynamics, and transport. In parallel, cytoskeletal researchers have revealed a fantastic diversity of structure and cellular function in both actin and microtubules. Increasingly, these areas overlap, necessitating an understanding of both organelle and cytoskeletal biology. This review addresses connections between the actin/microtubule cytoskeletons and organelles in animal cells, focusing on three key areas: ER structure and function; ER-to-Golgi transport; and Golgi structure and function. Making these connections has been challenging for several reasons: the small sizes and dynamic characteristics of some components; the fact that organelle-specific cytoskeletal elements can easily be obscured by more abundant cytoskeletal structures; and the difficulties in imaging membranes and cytoskeleton simultaneously, especially at the ultrastructural level. One major concept is that the cytoskeleton is frequently used to generate force for membrane movement, with two potential consequences: translocation of the organelle, or deformation of the organelle membrane. While initially discussing issues common to metazoan cells in general, we subsequently highlight specific features of neurons, since these highly polarized cells present unique challenges for organellar distribution and dynamics
Novel Roles for Actin in Mitochondrial Fission
Mitochondrial dynamics, including fusion, fission and translocation, are crucial to cellular homeostasis, with roles in cellular polarity, stress response and apoptosis. Mitochondrial fission has received particular attention, owing to links with several neurodegenerative diseases. A central player in fission is the cytoplasmic dynamin-related GTPase Drp1, which oligomerizes at the fission site and hydrolyzes GTP to drive membrane ingression. Drp1 recruitment to the outer mitochondrial membrane (OMM) is a key regulatory event, which appears to require a pre-constriction step in which the endoplasmic reticulum (ER) and mitochondrion interact extensively, a process termed ERMD (ER-associated mitochondrial division). It is unclear how ER-mitochondrial contact generates the force required for pre-constriction or why pre-constriction leads to Drp1 recruitment. Recent results, however, show that ERMD might be an actin-based process in mammals that requires the ER-associated formin INF2 upstream of Drp1, and that myosin II and other actin-binding proteins might be involved. In this Commentary, we present a mechanistic model for mitochondrial fission in which actin and myosin contribute in two ways; firstly, by supplying the force for pre-constriction and secondly, by serving as a coincidence detector for Drp1 binding. In addition, we discuss the possibility that multiple fission mechanisms exist in mammals
Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering
The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor
delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic
protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with
BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh
scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4%
concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4–
PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were
seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the
initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had
similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third
week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation
within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation
on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but
this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed
a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single,
simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in
vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher
with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher
ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers
and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP
activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth
factors holds promise for the production of tissue engineered bone.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)
Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration
The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)
3D plotted PCL scaffolds for stem cell based bone tissue engineering
The ability to control the architecture and strength of a bone tissue
engineering scaffold is critical to achieve a harmony between the scaffold and the
host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to
satisfy this need and to create a scaffold directly from the scanned and digitized
image of the defect site. Design and construction of complex structures with
different shapes and sizes, at micro and macro scale, with fully interconnected pore
structure and appropriate mechanical properties are possible by using RP techniques.
In this study, RP was used for the production of poly(e-caprolactone) (PCL) scaffolds.
Scaffolds with four different architectures were produced by using different configurations
of the fibers (basic, basic-offset, crossed and crossed-offset) within the
architecture of the scaffold. The structure of the prepared scaffolds were examined by
scanning electron microscopy (SEM), porosity and its distribution were analyzed by
micro-computed tomography (m-CT), stiffness and modulus values were determined
by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very
ordered structures with mean porosities about 60%, and having storage modulus
values about 1!107 Pa. These structures were then seeded with rat bone marrow
origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold
structure on the cell behavior; the proliferation and differentiation of the cells on
the scaffolds were studied. It was observed that cell proliferation was higher on offset
scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and
stainings for actin filaments of the cells reveal successful attachment and spreading
at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher
for the samples with lower cell proliferation, as expected. Highest MSC differentiation
was observed for crossed scaffolds indicating the influence of scaffold structure on
cellular activities
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Tissue-type plasminogen activator-primed human iPSC-derived neural progenitor cells promote motor recovery after severe spinal cord injury.
The goal of stem cell therapy for spinal cord injury (SCI) is to restore motor function without exacerbating pain. Induced pluripotent stem cells (iPSC) may be administered by autologous transplantation, avoiding immunologic challenges. Identifying strategies to optimize iPSC-derived neural progenitor cells (hiNPC) for cell transplantation is an important objective. Herein, we report a method that takes advantage of the growth factor-like and anti-inflammatory activities of the fibrinolysis protease, tissue plasminogen activator tPA, without effects on hemostasis. We demonstrate that conditioning hiNPC with enzymatically-inactive tissue-type plasminogen activator (EI-tPA), prior to grafting into a T3 lesion site in a clinically relevant severe SCI model, significantly improves motor outcomes. EI-tPA-primed hiNPC grafted into lesion sites survived, differentiated, acquired markers of motor neuron maturation, and extended βIII-tubulin-positive axons several spinal segments below the lesion. Importantly, only SCI rats that received EI-tPA primed hiNPC demonstrated significantly improved motor function, without exacerbating pain. When hiNPC were treated with EI-tPA in culture, NMDA-R-dependent cell signaling was initiated, expression of genes associated with stemness (Nestin, Sox2) was regulated, and thrombin-induced cell death was prevented. EI-tPA emerges as a novel agent capable of improving the efficacy of stem cell therapy in SCI
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