TCL/RhoJ is a Cdc42-related Rho GTPase with reported activities in endothelial cell biology and angiogenesis, metastatic melanoma, and corneal epithelial cells; however, less is known about how it is inherently regulated in comparison to its closest homologues TC10 and Cdc42. TCL has an N-terminal extension of 18 amino acids in comparison to Cdc42, but the function of this amino acid sequence has not been elucidated. A truncation mutant lacking the N terminus (ΔN) was found to alter TCL plasma membrane localization and nucleotide binding, and additional truncation and point mutants mapped the alterations of TCL biochemistry to amino acids 17-20. Interestingly, whereas the TCL ΔN mutant clearly influenced nucleotide exchange, deletion of the N terminus from its closest homologue, TC10, did not have a similar effect. Chimeras of TCL and TC10 revealed amino acids 121-129 of TCL contributed to the differences in nucleotide loading. Together, these results identify amino acids within the N terminus and a loop region distal to the nucleotide binding pocket of TCL capable of allosterically regulating nucleotide exchange and thus influence membrane association of the protein.

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the ‘gold standard’ for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology.

Using DNA ligase‐catalyzed cyclization assays, the in vitro persistence length of DNA has been determined to be about 150 base pairs (bp). In living E. coli bacteria, where DNA loops regulate the lac operon, DNA looping appears to be much more probable than expected from in vitro data. Why? A possible explanation for this paradox is that DNA loops in bacteria are bridged by proteins, reducing the amount of required costly DNA bending. Current in vitro methods to measure DNA flexibility are tedious and artificial, in that they require DNA cyclization, not just DNA looping. No high‐throughput method has been described to measure DNA flexibility for loops involving bridging proteins of different sizes. We are developing a high‐throughput method to measure the probability of DNA looping when bridged by a fusion protein that includes PCV2 and micrococcal nuclease domains. A bead‐tethered 1000‐bp DNA molecule of a known sequence is conjugated to this fusion protein through the PCV2 domain, and looping allows self‐cleavage such that the distribution of cleavage sites revealed by next generation sequencing reflects the energetics of DNA looping. We hypothesize that this distribution will be affected as a function of bridging protein size. Measuring how the size of bridging proteins affects tight DNA loop probability enables improved understanding of genetic switches.

TCL (RhoJ) belongs to the Rho family of GTPases, and while it has been shown to contribute to angiogenesis and metastatic melanoma, less is known about its molecular and cellular function. To better understand the inherent cellular biochemistry of TCL, our lab has been performing a detailed structure/function analysis of the GTPase. Previously, the extended N terminus of TCL was found to be important for GTP‐loading and localization to the plasma membrane. The deletion or mutation of this TCL‐specific sequence led to inactivation of TCL and its localizing to intracellular vesicles. To determine if the N‐terminus of TCL contributes to cellular localization by itself, expression plasmids were produced where the first 24 N‐terminal amino acids and last 21 C‐terminal amino acids of TCL were fused to the N‐ and C‐termini (respectively) of the fluorescent protein Venus. Interestingly, expression of the constructs bearing only the C‐terminal tail of TCL or both the N‐ and C‐termini sequestered Venus to the plasma membrane, indicating the C‐terminal tail is potentially sufficient for TCL membrane localization. Additionally, experiments using constitutively active (CA) and dominant negative (DN) mutants showed CA TCL localized to the plasma membrane while DN TCL was vesicular; however, CA TCL where C terminus has been deleted results in diffuse cytoplasmic localization, while the same deletions in the context of the DN mutation lead to a marked vesicular localization. Together, our results suggest TCL is specifically retained to vesicles through an interaction with an as yet unidentified binding partner, and future experiments will use biotinligase tags of CA and DN TCL and BioID procedures to determine what hypothetical partner protein is retaining TCL to vesicular membranes.Support or Funding InformationSupport was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.