Limits...
Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking.

Richards TS, Knowlton AE, Grieshaber SS - BMC Microbiol. (2013)

Bottom Line: The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins.The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion.Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized.

Results: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs.

Conclusions: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Show MeSH

Related in: MedlinePlus

Inclusion fusion is delayed in cells with multiple unclustered centrosomes. HeLa cells (A) and neuroblastomas (B) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ27 and fixed at 3 and 24 hpi. Cells were stained with anti-g-tubulin antibodies (green) and human sera (red). HeLa cells (C) and neuroblastomas (D) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ3, 9 and 27 and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and inclusions were enumerated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750546&req=5

Figure 4: Inclusion fusion is delayed in cells with multiple unclustered centrosomes. HeLa cells (A) and neuroblastomas (B) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ27 and fixed at 3 and 24 hpi. Cells were stained with anti-g-tubulin antibodies (green) and human sera (red). HeLa cells (C) and neuroblastomas (D) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ3, 9 and 27 and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and inclusions were enumerated.

Mentions: We established that inclusion fusion occurs at cell centrosomes and both dynein and microtubules promote fusion. We next asked whether infection of cells with multiple centrosomes would lead to multiple sites of fusion. The mouse neuroblastoma cell line N115 has significant centrosome number defects containing an average of eight centrosomes per cell [13,14]. This allowed us to ask whether defects in centrosome numbers would affect inclusion fusion. HeLa and neuroblastoma cells were infected with C. trachomatis at three different multiplicities of infection. Infections were fixed at 3 hpi and every two hours between 10 and 24 hpi. Early inclusions were present near the tightly clustered centrosomes in HeLa cells but in neuroblastoma cells, which have multiple centrosomes distributed throughout the cell, early inclusions were present throughout the host cytosol clustered at the scattered centrosomes (Figure 4A 3 hpi and 4B 3 hpi, respectively). At 24 hpi, infected HeLa cells had a single inclusion adjacent to the centrosomes (Figure 4 24 hpi). While some infected neuroblastoma cells had single inclusions at 24 hpi, infected neuroblastoma cells could still be found with multiple unfused inclusions (Figure 4B 24 hpi). In infected HeLa cells, fusion of chlamydial inclusions occurred at approximately 12-14 hpi (Figure 4C). Fusion was delayed in neuroblastoma cells, occurring at approximately 16-18 hpi (Figure 4D).


Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking.

Richards TS, Knowlton AE, Grieshaber SS - BMC Microbiol. (2013)

Inclusion fusion is delayed in cells with multiple unclustered centrosomes. HeLa cells (A) and neuroblastomas (B) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ27 and fixed at 3 and 24 hpi. Cells were stained with anti-g-tubulin antibodies (green) and human sera (red). HeLa cells (C) and neuroblastomas (D) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ3, 9 and 27 and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and inclusions were enumerated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750546&req=5

Figure 4: Inclusion fusion is delayed in cells with multiple unclustered centrosomes. HeLa cells (A) and neuroblastomas (B) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ27 and fixed at 3 and 24 hpi. Cells were stained with anti-g-tubulin antibodies (green) and human sera (red). HeLa cells (C) and neuroblastomas (D) were infected with C. trachomatis at MOI‚ÄČ~‚ÄČ3, 9 and 27 and fixed at 10, 12, 14, 16, 20, 22 and 24 hpi. Cells were stained with human sera and inclusions were enumerated.
Mentions: We established that inclusion fusion occurs at cell centrosomes and both dynein and microtubules promote fusion. We next asked whether infection of cells with multiple centrosomes would lead to multiple sites of fusion. The mouse neuroblastoma cell line N115 has significant centrosome number defects containing an average of eight centrosomes per cell [13,14]. This allowed us to ask whether defects in centrosome numbers would affect inclusion fusion. HeLa and neuroblastoma cells were infected with C. trachomatis at three different multiplicities of infection. Infections were fixed at 3 hpi and every two hours between 10 and 24 hpi. Early inclusions were present near the tightly clustered centrosomes in HeLa cells but in neuroblastoma cells, which have multiple centrosomes distributed throughout the cell, early inclusions were present throughout the host cytosol clustered at the scattered centrosomes (Figure 4A 3 hpi and 4B 3 hpi, respectively). At 24 hpi, infected HeLa cells had a single inclusion adjacent to the centrosomes (Figure 4 24 hpi). While some infected neuroblastoma cells had single inclusions at 24 hpi, infected neuroblastoma cells could still be found with multiple unfused inclusions (Figure 4B 24 hpi). In infected HeLa cells, fusion of chlamydial inclusions occurred at approximately 12-14 hpi (Figure 4C). Fusion was delayed in neuroblastoma cells, occurring at approximately 16-18 hpi (Figure 4D).

Bottom Line: The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins.The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion.Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized.

Results: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs.

Conclusions: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Show MeSH
Related in: MedlinePlus