Beyond the Matrix: The Many Non-ECM Ligands for Integrins.

The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM), and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins.

Notch: A multi-functional integrating system of microenvironmental signals.

The Notch signaling cascade is an evolutionarily ancient system that allows cells to interact with their microenvironmental neighbors through direct cell-cell interactions, thereby directing a variety of developmental processes. Recent research is discovering that Notch signaling is also responsive to a broad variety of stimuli beyond cell-cell interactions, including: ECM composition, crosstalk with other signaling systems, shear stress, hypoxia, and hyperglycemia. Given this emerging understanding of Notch responsiveness to microenvironmental conditions, it appears that the classical view of Notch as a mechanism enabling cell-cell interactions, is only a part of a broader function to integrate microenvironmental cues. In this review, we summarize and discuss published data supporting the idea that the full function of Notch signaling is to serve as an integrator of microenvironmental signals thus allowing cells to sense and respond to a multitude of conditions around them.

Notch intracellular domains form transcriptionally active heterodimeric complexes on sequence-paired sites.

Notch signaling is universally conserved in metazoans where it is important for a wide variety of both normal and abnormal physiology. All four mammalian Notch receptors are activated by a conserved mechanism that releases Notch intracellular domains (NICDs) from the plasma membrane to translocate to the nucleus. Once there, NICDs interact through highly conserved ankyrin domains to form head-to-head homodimers on Notch sensitive promoters and stimulate transcription. Due to the highly conserved nature of these Notch ankyrin domains in all four mammalian Notch proteins, we hypothesized that NICDs may also engage in heterodimerization. Our results reveal the presence of two NICD dimerization states that can both engage in homo and heterodimerization. Using a Co-IP approach, we show that all NICD's can form non-transcriptionally active dimers and that the N4ICD appears to perform this function better than the other NICDs. Using a combination of ChIP analysis and transcriptional reporter assays, we also demonstrate the formation of transcriptionally active heterodimers that form on DNA. In particular, we demonstrate heterodimerization between the N2ICD and N4ICD and show that this heterodimer pair appears to exhibit differential activity on various Notch sensitive promoters. These results illustrate a new diversification of Notch signaling mechanisms which will help us better understand basic Notch function.

Notch Intracellular Domains form Transcriptionally Active Heterodimeric Complexes on Sequence-paired Sites.

Notch signaling is universally conserved in metazoans where it is important for a wide variety of both normal and abnormal physiology. All four mammalian Notch receptors are activated by a conserved mechanism that releases Notch intracellular domains (NICDs) from the plasma membrane to translocate to the nucleus. Once there, NICDs interact through highly conserved ankyrin domains to form head-to-head homodimers on Notch sensitive promoters and stimulate transcription. Due to the highly conserved nature of these Notch ankyrin domains in all four mammalian Notch proteins, we hypothesized that NICDs may also engage in heterodimerization. Our results reveal the presence of two NICD dimerization states that can both engage in homo and heterodimerization. Using a Co-IP approach, we show that all NICD's can form non-transcriptionally active dimers and that the N4ICD appears to perform this function better than the other NICDs. Using a combination of ChIP analysis and transcriptional reporter assays, we also demonstrate the formation of transcriptionally active heterodimers that form on DNA. In particular, we demonstrate heterodimerization between the N2ICD and N4ICD and show that this heterodimer pair appears to exhibit differential activity on various Notch sensitive promoters. These results illustrate a new diversification of Notch signaling mechanisms which will help us better understand basic Notch function.

Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites.

Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch's ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members' SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.