EGFR family and Src family kinase interactions: mechanics matters?

Receptor tyrosine kinases (RTKs), such as the EGF receptor family, and adhesion molecules, such as integrins, have historically been viewed to have distinctly separable roles in the cell. In this classical view, integrins mediate mechanical interactions between the cell and its surrounding extracellular matrix while RTKs handle signaling to modulate cellular behavior. Although crosstalk between these receptor pathways has been known to exist for a long time, this has generally been attributed to effects significantly downstream from the receptors themselves. In recent years, however, EGFR family RTKs have been found to directly participate in integrin-mediated force sensing, revealing a more complex interplay among these cellular components than originally appreciated. Here we briefly review the classical understanding of EGFR family RTK signaling and then provide a broadened perspective based on recent results.

Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.

Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.

Reconstitution of microtubule-dependent organelle transport.

Microtubule (MT)-based motor proteins transport many cellular factors to their functionally relevant locations within cells, and defects in transport are linked to human disease. Understanding the mechanism and regulation of this transport process in living cells is difficult because of the complex in vivo environment and limited means to manipulate the system. On the other hand, in vitro motility assays using purified motors attached to beads does not recapitulate the full complexity of cargo transport in vivo. Assaying motility of organelles in cell extracts is therefore attractive, as natural cargoes are being examined, but in an environment that is more amenable to manipulation. Here, we describe the purification and in vitro MT-based motility of phagosomes from Dictyostelium and lipid droplets from rat liver. These assays have the potential to address diverse questions related to endosome/phagosome maturation, fatty acid regulation, and could also serve as a starting point for reconstituting the motility of other types of organelles.

HindIII-based restriction fragment length polymorphism in hemophilic and nonhemophilic patients.

Hemophilia A is most common recessively inherited bleeding disorder, which affect one in five thousand male births throughout the world. In most of the hemophilic A patients, no common mutation is easily identifiable. This limitation has been overcome by the use of polymorphic DNA marker, i.e., restriction fragment length polymorphism (RFLP). This marker of polymorphism could only be detected by amplifying the polymorphic region and digestion the polymerase chain reaction (PCR) product with the restriction enzyme (PCR-RFLP), i.e., HindIII. The polymorphic region of HindIII is 608 bp in length and after the restriction digestion, different sizes of fragments, i.e., 427 and 181 bp were, respectively, obtained. However, in homozygous (+/+) condition three bands of 427, 100, and 81 bp were obtained and in the other negative allelic homozygous condition (-/-) two bands of 427 and 181 bp were obtained. Similarly fragments of different sizes, i.e., 427, 181, 100, and 81 bp were obtained in heterozygous conditions. Therefore, in this study, we have analyzed the factor VIII gene in the 17 different families using restriction enzyme HindIII-based RFLP molecular marker technique. Out of these, the observed heterozygosity for HindIII was found 47.5%, whereas, for positive allele it was 26%, and for negative allele the frequency was 74%.