Transcriptional profiling of Dictyostelium development reveals significant conservation of transcriptional profiles between evolutionarily divergent species.

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.

Terminase enzyme complexes, which facilitate ATP-driven DNA packaging in phages and in many eukaryotic viruses, constitute a wide and potentially diverse family of molecular motors about which little dynamic or mechanistic information is available. Here we report optical tweezers measurements of single DNA molecule packaging dynamics in phage T4, a large, tailed Escherichia coli virus that is an important model system in molecular biology. We show that a complex is formed between the empty prohead and the large terminase protein (gp17) that can capture and begin packaging a target DNA molecule within a few seconds, thus demonstrating a distinct viral assembly pathway. The motor generates forces >60 pN, similar to those measured with phage 29, suggesting that high force generation is a common property of viral DNA packaging motors. However, the DNA translocation rate for T4 was strikingly higher than that for 29, averaging Ϸ700 bp/s and ranging up to Ϸ2,000 bp/s, consistent with packaging by phage T4 of an enormous, 171-kb genome in <10 min during viral infection and implying high ATP turnover rates of >300 s ؊1 . The motor velocity decreased with applied load but averaged 320 bp/s at 45 pN, indicating very high power generation. Interestingly, the motor also exhibited large dynamic changes in velocity, suggesting that it can assume multiple active conformational states gearing different translocation rates. This capability, in addition to the reversible pausing and slipping capabilities that were observed, may allow phage T4 to coordinate DNA packaging with other ongoing processes, including viral DNA transcription, recombination, and repair.bacteriophage T4 ͉ molecular motor ͉ optical tweezers ͉ single-molecule ͉ viral DNA packaging A critical step in the assembly of many viruses is the packaging of the viral genome into a preassembled prohead shell by the action of an ATP-powered molecular motor (1, 2). Systems in which this mode of assembly occurs include numerous tailed dsDNA and dsRNA phages and certain animal viruses, including adenoviruses and herpesviruses. Viral DNA packaging complexes thus constitute a wide and potentially diverse family of molecular motors that are considerably understudied compared with cellular molecular motors such as myosins, kinesins, and helicases.In a typical phage assembly pathway, a prohead shell of precise dimensions co-assembles with a scaffolding core. One of the vertices of the prohead is unique, containing a dodecameric portal ring structure (3). When the scaffolding leaves, a defined space is created inside the capsid. A packaging ATPase complex then docks onto the outer end of the portal, inserting one end of the viral genome into the 3.5-to 4-nm channel, and translocates the DNA by using ATP hydrolysis energy (2, 4). After genome packaging, the ATPase dissociates, leaving the portal with the head, the outer surface of which provides a platform for the assembly of tail components. When the virus infects a cell, the densely packed DNA exits rapidly through the portal ch...

Molecular motors drive genome packaging into preformed procapsids in many dsDNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage λ. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage ϕ29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ~600 bp/s, which is 3.5-fold higher than ϕ29, and the motor processivity was also 3-fold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging in many cases, and greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid.

Chemotaxis, the chemically guided movement of cells, plays an important role in several biological processes including cancer, wound healing, and embryogenesis. Chemotacting cells are able to sense shallow chemical gradients where the concentration of chemoattractant differs by only a few percent from one side of the cell to the other, over a wide range of local concentrations. Exactly what limits the chemotactic ability of these cells is presently unclear. Here we determine the chemotactic response of Dictyostelium cells to exponential gradients of varying steepness and local concentration of the chemoattractant cAMP. We find that the cells are sensitive to the steepness of the gradient as well as to the local concentration. Using information theory techniques, we derive a formula for the mutual information between the input gradient and the spatial distribution of bound receptors and also compute the mutual information between the input gradient and the motility direction in the experiments. A comparison between these quantities reveals that for shallow gradients, in which the concentration difference between the back and the front of a 10-μm-diameter cell is <5%, and for small local concentrations (<10 nM) the intracellular information loss is insignificant. Thus, external fluctuations due to the finite number of receptors dominate and limit the chemotactic response. For steeper gradients and higher local concentrations, the intracellular information processing is suboptimal and results in a smaller mutual information between the input gradient and the motility direction than would have been predicted from the ligand-receptor binding process.mutual information | noise limits | Dictyostelium | microfluidics C hemotaxis, the motion of cells guided by chemical gradients, plays an important role in a variety of biological processes, including wound healing, embryogenesis, and cancer metastasis. The chemical gradients required for efficient chemotaxis can be very shallow for eukaryotic cells. For example, the rapidly crawling neutrophils of the mammalian immune system and the social amoebae, Dictyostelium discoideum (1-8), are able to sense shallow chemical gradients where the concentration of chemoattractant differs by only a few percent from one side of the cell to the other, over a wide range of local concentrations (9-11).The chemotactic response of these cells can be considered as the outcome from two distinct steps: establishment of spatial differences in the distribution of receptors with bound chemoattractant on the cell's surface (12) and the response to these differences by the signal transduction pathways leading to directed motility (13). The first step is subject to the external fluctuations in chemoattractant binding to the surface receptor. This external noise can be precisely characterized, either through direct numerical simulations (14, 15) or through approximate analytical calculations (16-18). The second step involves a number of pathways that are subject to internal background noise generated ...