Molecular Biology of the Cell
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Published By American Society For Cell Biology

1939-4586, 1059-1524

Author(s):  
Benjamin W. Scandling ◽  
Jia Gou ◽  
Jessica Thomas ◽  
Jacqueline Xuan ◽  
Chuan Xue ◽  
...  

Many cells in the body experience cyclic mechanical loading, which can impact cellular processes and morphology. In vitro studies often report that cells reorient in response to cyclic stretch of their substrate. To explore cellular mechanisms involved in this reorientation, a computational model was developed by utilizing the previous computational models of the actin-myosin-integrin motor-clutch system developed by others. The computational model predicts that under most conditions, actin bundles align perpendicular to the direction of applied cyclic stretch, but under specific conditions, such as low substrate stiffness, actin bundles align parallel to the direction of stretch. The model also predicts that stretch frequency impacts the rate of reorientation, and that proper myosin function is critical in the reorientation response. These computational predictions are consistent with reports from the literature and new experimental results presented here. The model suggests that the impact of different stretching conditions (stretch type, amplitude, frequency, substrate stiffness, etc.) on the direction of cell alignment can largely be understood by considering their impact on cell-substrate detachment events, specifically whether detachment occurs during stretching or relaxing of the substrate.


Author(s):  
Roberta B. Nowak ◽  
Haleh Alimohamadi ◽  
Kersi Pestonjamasp ◽  
Padmini Rangamani ◽  
Velia M. Fowler

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ is not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule STORM imaging of Alexa-647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, non-random distribution of F-actin nodes. Treatment of RBCs with LatA and CytoD indicates F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape and membrane deformability.


Author(s):  
Junya Hasegawa ◽  
Emi Tokuda ◽  
Yao Yao ◽  
Takehiko Sasaki ◽  
Ken Inoki ◽  
...  

Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of TFEB at multiple sites including Ser-211. Here we show that inhibition of PIKfyve, which produces phosphatidylinositol 3,5-bisphosphate on endosomes and lysosomes, causes a loss of Ser-211 phosphorylation and concomitant nuclear localization of TFEB. We found that while mTORC1 activity toward S6K1, as well as other major mTORC1 substrates, is not impaired, PIKfyve inhibition specifically impedes the interaction of TFEB with mTORC1. This suggests that mTORC1 activity on TFEB is selectively inhibited due to loss of mTORC1 access to TFEB. In addition, we found that TFEB activation during inhibition of PIKfyve relies on the ability of protein phosphatase 2A (PP2A) but not calcineurin/PPP3, to dephosphorylate TFEB Ser-211. Thus, when PIKfyve is inhibited, PP2A is dominant over mTORC1 for control of TFEB phosphorylation at Ser-S211. Together these findings suggest that mTORC1 and PP2A have opposing roles on TFEB via phosphorylation and dephosphorylation of Ser-211, respectively, and further, that PIKfyve inhibits TFEB activity by facilitating mTORC1-dependent phosphorylation of TFEB.


Author(s):  
Carlo Giovanni Quintanilla ◽  
Wan-Ru Lee ◽  
Jen Liou

Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) in receptor-stimulated cells is mediated by the lipid transfer protein Nir2. Nir2 is dynamically recruited to endoplasmic reticulum-plasma membrane (ER-PM) junctions to facilitate replenishment of PM PIP2 hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP2 is limited. Here, we describe the functions of Nir1 as a positive regulator of Nir2 and PIP2 homeostasis. In contrast to the family proteins Nir2 and Nir3, Nir1 constitutively localizes at ER-PM junctions. Nir1 potentiates Nir2 targeting to ER-PM junctions during receptor-mediated signaling and is required for efficient PM PIP2 replenishment. Live-cell imaging and biochemical analysis reveal that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Combined, results from this study identify Nir1 as an ER-PM junction localized protein that promotes Nir2 recruitment for PIP2 homeostasis.


Author(s):  
Rafael E. Sanchez-Pupo ◽  
Brooke L. O'Donnell ◽  
Danielle Johnston ◽  
Laszlo Gyenis ◽  
David W. Litchfield ◽  
...  

Pannexins (PANX) are a family of three channel-forming membrane glycoproteins expressed in the skin. Previous studies have focused on the role of PANX1 and PANX3 in the regulation of cellular functions in skin cells while PANX2, the largest member of this protein family, has not been investigated. In the current study, we explored the temporal PANX2 expression in murine skin and found that one Panx2 splice variant ( Panx2-202) tends to be more abundant at the protein level and is continuously expressed in developed skin. PANX2 was detected in the suprabasal layers of the mouse epidermis and upregulated in an in vitro model of rat epidermal keratinocyte differentiation. Furthermore, we showed that in apoptotic rat keratinocytes, upon UVB-induced caspase-3/7 activation, ectopically overexpressed PANX2 is cleaved in its C-terminal domain at D416 residue without increasing the apoptotic rate measured by caspase-3/7 activation. Notably, CRISPR-Cas9-mediated genetic deletion of rat Panx2 delays but does not impair caspase-3/7 activation and cytotoxicity in UVB-irradiated keratinocytes. We propose that endogenous PANX2 expression in keratinocytes promotes cell death after UVB insult and may contribute to skin homeostasis.


Author(s):  
Heidi L. Anderson ◽  
Jason C. Casler ◽  
Laura L. Lackner

Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.


Author(s):  
Apratim Mukherjee ◽  
Haonan Zhang ◽  
Katherine Ladner ◽  
Megan Brown ◽  
Jacob Urbanski ◽  
...  

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous sub-mesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here, we fabricated extracellular-matrix mimicking suspended fiber networks: crosshatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying inter-fiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease, while force-disease biphasic relationship exhibited f-actin stress-fiber network dependence. However, unique to suspended fibers, coiling occurring at tips of protrusions and not the length or breadth of protrusions displayed strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on time scale of hours. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]


Author(s):  
Amanjot Singh ◽  
Arvind Reddy Kandi ◽  
Deepa Jayaprakashappa ◽  
Guillaume Thuery ◽  
Devam J Purohit ◽  
...  

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3-hours predominantly resulted in induction or upregulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for chaperones, including HSP70s, small heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.


2022 ◽  
Vol 33 (1) ◽  
Author(s):  
Donna K. Ginther

It has been a decade since “Race, Ethnicity, and NIH Research Awards” was published. Receiving the American Society for Cell Biology Public Service Award allows me to reflect on this research and its impact. In this essay, I share the story of how my research interests and professional networks provided the opportunity to do this important work. I also make the case for improved data and mentoring to address race and ethnic disparities in NIH funding.


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